Double-acylated GLP-1 derivatives

ABSTRACT

The invention relates to a derivative of a GLP-1 analogue, which analogue comprises a first K residue at a position corresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1), a second K residue at a position corresponding to position 26 of GLP-1(7-37), and a maximum of ten amino acid modifications as compared to GLP-1(7-37), wherein the first K residue is designated K 37 , and the second K residue is designated K 26 , which derivative comprises two albumin binding moieties attached to K 26  and K 37 , respectively, wherein the albumin binding moiety comprises a protracting moiety selected from: 
     Chem. 1, Chem. 2, Chem. 3 or Chem. 4; or a pharmaceutically acceptable salt, amide, or ester thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. 119 of Danishapplication EP09179390.1 filed Dec. 16, 2009, and of U.S. provisionalapplication 61/288,601, filed Dec. 21, 2009, the contents of which arehereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to derivatives of Glucagon-Like Peptide 1(GLP-1) and their pharmaceutical use, viz. to double-acylated GLP-1derivatives acylated at position 26 and 37, and their pharmaceuticaluse.

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

In accordance with 37 C.F.R. §1.52(e)(5), Applicants enclose herewiththe Sequence Listing for the above-captioned application entitled“SEQUENCE LISTING”, created on Jun. 26, 2013. The Sequence Listing ismade up of 6 kilobytes, and the information contained in the attached“SEQUENCE LISTING” is identical to the information in the specificationas originally filed. No new matter is added.

BACKGROUND OF THE INVENTION

Journal of Medicinal Chemistry (2000), vol. 43, no. 9, p. 1664-669discloses derivatives of GLP-1(7-37) that are double-acylated atK^(26,34)-see Table 1.

WO 98/08871 discloses a number of GLP-1 derivatives including some thatare double-acylated at K^(26,34), see Examples 3, 7, 17, 24, 32, 33, and36. Liraglutide, a mono-acylated GLP-1 derivative for once dailyadministration which is marketed as of 2009 by Novo Nordisk A/S, is alsodisclosed in WO 98/08871 (Example 37). WO 99/43706 discloses a number ofmono- and double-acylated GLP-1 derivatives including some K^(26,37)derivatives (see p. 148-178).

WO 2005/027978 discloses a number of GLP-1 derivatives including a fewthat are double-acylated at one and the same residue, K³⁷, see Examples8 and 9.

WO 2009/030738 discloses a number of GLP-1 derivatives including onedouble-acylated at K³¹, Dap³⁴, see Example 37.

Journal of Controlled Release (2010), vol. 144, p. 10-16 relates toacylated exendin-4 analogs and discloses, among others, adouble-acylated exendin-4 (K^(12,27)-diLUA-Exendin-4) is disclosed (LUAis lauric acid, C12).

WO 06/097537 discloses a number of GLP-1 derivatives includingsemaglutide (Example 4), a mono-acylated GLP-1 derivative for onceweekly administration which is under development by Novo Nordisk A/S.

Angewandte Chemie International Edition 2008, vol. 47, p. 3196-3201reports the discovery and characterisation of a class of4-(p-iodophenyl)butyric acid derivatives which purportedly display astable noncovalent binding interaction with both mouse serum albumin(MSA) and human serum albumin (HSA).

SUMMARY OF THE INVENTION

The invention relates to derivatives of GLP-1 peptides.

The derivatives are acylated at the native lysine at position 26, aswell as at a lysine substituted for the native glycine at position 37.The side chains are albumin binding moieties. They comprise aprotracting moiety, preferably selected from fatty diacids, and fattyacids with a distal phenyl, phenoxy, or thiophene group, all optionallysubstituted. A carboxy group of the fatty acid or fatty diacid isacylated, optionally via a linker, to a lysine residue of the GLP-1peptide, preferably at the epsilon-amino group thereof. The GLP-1peptide may be an analogue of GLP-1(7-37) (SEQ ID NO: 1) having a totalof up to ten amino acid differences as compared to GLP-1(7-37), forexample one or more additions, one or more deletions, and/or one or moresubstitutions.

More in particular, the invention relates to a derivative of a GLP-1analogue, which analogue comprises a first K residue at a positioncorresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1), a second Kresidue at a position corresponding to position 26 of GLP-1(7-37), and amaximum of ten amino acid modifications as compared to GLP-1(7-37),wherein the first K residue is designated K³⁷, and the second K residueis designated K²⁶; which derivative comprises two albumin bindingmoieties attached to K²⁶ and K³⁷, respectively, wherein each albuminbinding moiety comprises a protracting moiety selected from Chem. 1,Chem. 2, Chem. 3, and Chem. 4:HOOC—(CH₂)_(x)—CO—*  Chem. 1:HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:R¹—C₆H₄—(CH₂)_(z)—CO—*  Chem. 3:HOOC—C₄SH₂—(CH₂)_(w)—CO—*,  Chem. 4:in which x is an integer in the range of 6-18, y is an integer in therange of 3-17, z is an integer in the range of 1-5, R¹ is a group havinga molar mass not higher than 150 Da, and w is an integer in the range of6-18; with the proviso that when the protracting moiety is Chem. 1, thealbumin binding moiety further comprises a linker of formula Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5; or a pharmaceutically acceptable salt, amide, or esterthereof.

The invention also relates to such derivative for use as a medicament,in particular for use in the treatment and/or prevention of all forms ofdiabetes and related diseases, such as eating disorders, cardiovasculardiseases, gastrointestinal diseases, diabetic complications, criticalillness, and/or polycystic ovary syndrome; and/or for improving lipidparameters, improving β-cell function, and/or for delaying or preventingdiabetic disease progression.

The invention furthermore relates to intermediate products in the formof GLP-1 peptides and side chains, which are relevant for thepreparation of certain GLP-1 peptides and derivatives of the invention.

The derivatives of the invention are biologically active. Also, oralternatively, they have a protracted pharmacokinetic profile. Also, oralternatively, they are stable against degradation by gastro intestinalenzymes. Also, or alternatively, they have a high oral bioavailability.These properties are of importance in the development of next generationGLP-1 compounds for subcutaneous, intravenous, and/or in particular oraladministration.

DESCRIPTION OF THE INVENTION

The invention relates to derivatives of GLP-1 peptides. The derivativesare acylated at the native lysine at position 26, as well as at a lysinesubstituted for the native glycine at position 37. The side chains arealbumin binding moieties. They comprise a protracting moiety, preferablyselected from fatty diacids, and fatty acids with a distal, or terminal,phenyl, thiophene, or phenoxy group, all optionally substituted. Acarboxy group of the fatty acid or fatty diacid is acylated, optionallyvia a linker, to a lysine residue of the GLP-1 peptide, preferably atthe epsilon-amino group thereof. The GLP-1 peptide may be an analogue ofGLP-1(7-37) (SEQ ID NO: 1) having a total of up to ten amino aciddifferences as compared to GLP-1(7-37), for example one or moreadditions, one or more deletions, and/or one or more substitutions.

More in particular, in a first aspect, the invention relates to aderivative of a GLP-1 analogue, which analogue comprises a first Kresidue at a position corresponding to position 37 of GLP-1(7-37) (SEQID NO: 1), a second K residue at a position corresponding to position 26of GLP-1(7-37), and a maximum of ten amino acid modifications ascompared to GLP-1(7-37), wherein the first K residue is designated K³⁷,and the second K residue is designated K²⁶, which derivative comprisestwo albumin binding moieties attached to K²⁶ and K³⁷, respectively,wherein the albumin binding moiety comprises a protracting moietyselected from Chem. 1, Chem. 2, Chem. 3, and Chem. 4:HOOC—(CH₂)_(x)—CO—*  Chem. 1:HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:R¹—C₆H₄—(CH₂)_(z)—CO—*  Chem. 3:HOOC—C₄SH₂—(CH₂)_(w)—CO—*  Chem. 4:in which x is an integer in the range of 6-18, y is an integer in therange of 3-17, z is an integer in the range of 1-5, R¹ is a group havinga molar mass not higher than 150 Da, and w is an integer in the range of6-18; with the proviso that when the protracting moiety is Chem. 1, thealbumin binding moiety further comprises a linker of formula Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5; or a pharmaceutically acceptable salt, amide, or esterthereof.

Thus, in a first aspect, the invention relates to a derivative of aGLP-1 analogue, wherein the GLP-1 analogue comprises a first K residueat a position corresponding to position 37 of GLP-1(7-37) (SEQ ID NO:1), a second K residue at a position corresponding to position 26 ofGLP-1(7-37), and a maximum of ten amino acid modifications as comparedto GLP-1(7-37), wherein the first K residue is designated K³⁷, and thesecond K residue is designated K²⁶, which derivative comprises twoalbumin binding moieties attached to K²⁶ and K³⁷, respectively, whereinthe albumin binding moiety comprises a protracting moiety selected fromChem. 2, Chem. 3, and Chem. 4:HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:R¹—C₆H₄—(CH₂)_(z)—CO—*  Chem. 3:HOOC—C₄SH₂—(CH₂)_(w)—CO—*  Chem. 4:in which y is an integer in the range of 3-17, z is an integer in therange of 1-5, R¹ is a group having a molar mass not higher than 150 Da,and w is an integer in the range of 6-18; or a pharmaceuticallyacceptable salt, amide, or ester thereof.

In a second aspect, the invention relates to a derivative of a GLP-1analogue, wherein the GLP-1 analogue comprises a first K residue at aposition corresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1), asecond K residue at a position corresponding to position 26 ofGLP-1(7-37), and a maximum of ten amino acid modifications as comparedto GLP-1(7-37), wherein the first K residue is designated K³⁷, and thesecond K residue is designated K²⁶, which derivative comprises twoalbumin binding moieties attached to K²⁶ and K³⁷, respectively, whereinthe albumin binding moiety comprises i) a protracting moiety of formulaChem. 1:HOOC—(CH₂)_(x)—CO—*  Chem. 1:in which x is an integer in the range of 6-18; and ii) a linker offormula Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5;or a pharmaceutically acceptable salt, amide, or ester thereof.

In a third aspect, the invention relates to a derivative of a GLP-1analogue, wherein the GLP-1 analogue comprises a first K residue at aposition corresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1), asecond K residue at a position corresponding to position 26 ofGLP-1(7-37), and a maximum of ten amino acid modifications as comparedto GLP-1(7-37), wherein the first K residue is designated K³⁷, and thesecond K residue is designated K²⁶; which derivative comprises twoprotracting moieties attached to K²⁶ and K³⁷, respectively, via alinker, wherein the protracting moiety is selected from Chem. 1, Chem.2, Chem. 3, and Chem. 4:HOOC—(CH₂)_(x)—CO—*  Chem. 1:HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:R¹—C₆H₄—(CH₂)_(z)—CO—*  Chem. 3:HOOC—C₄SH₂—(CH₂)_(w)—CO—*  Chem. 4:in which x is an integer in the range of 6-18, y is an integer in therange of 3-17, z is an integer in the range of 1-5, R¹ is a group havinga molar mass not higher than 150 Da, and w is an integer in the range of6-18; and the linker comprises Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5; or a pharmaceutically acceptable salt, amide, or esterthereof.

The invention also relates to an intermediate product in the form of aGLP-1 analogue which comprises the following modifications as comparedto GLP-1(7-37) (SEQ ID NO: 1): (i) (8Aib, 31H, 34Q, 37K); (ii) (des7-8,34R, 37K, 38E); (iii) (des7-8, 34R, 37K); (iv) (8Aib, 9G, 34R, 37K); (v)(8Aib, 23R, 34R, 37K); (vi) (31H, 34Q, 37K); (vii) (9Q, 34R, 37K); (iix)(30E, 34R, 37K); (ix) (34R, 37K, 38G); (x) (34R, 36G, 37K); or (xi)(34R, 37K, 38E); or a pharmaceutically acceptable salt, amide, or esterof any of the analogues thereof.

The invention also relates to an intermediate product comprising aprotracting moiety selected from Chem. 2c, Chem. 3b, and Chem. 4b:HOOC—C₆H₄—O—(CH₂)_(y)—CO—PG  Chem. 2c:R¹—C₆H₄—(CH₂)_(z)—CO—PG  Chem. 3b:HOOC—C₄H₂—(CH₂)_(w)—CO—PG  Chem. 4b:

in which y is an integer in the range of 3-17, z is an integer in therange of 1-5, R¹ is a group having a molar mass not higher than 150 Da,w is an integer in the range of 6-18, and *—CO—PG is an activated ester;wherein, optionally, the distal *—COOH group of the protracting moiety,if present, is functionalised as a non-reactive ester; or apharmaceutically acceptable salt, amide, or ester thereof.

And finally the invention also relates to the pharmaceutical use of theanalogues and derivatives of the invention, in particular for use in thetreatment and/or prevention of all forms of diabetes and relateddiseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression.

In what follows, Greek letters may be represented by their symbol or thecorresponding written name, for example: α=alpha; β=beta; ε=epsilon;γ=gamma; ω=omega; etc. Also, the Greek letter of μ my be represented by“u”, e.g. in μl=ul, or in μM=uM.

An asterisk (*) in a chemical formula designates i) a point ofattachment, ii) a radical, and/or iii) an unshared electron.

GLP-1 Analogues

The term “GLP-1 analogue” or “analogue of GLP-1” as used herein refersto a peptide, or a compound, which is a variant of the humanGlucagon-Like Peptide-1 (GLP-1(7-37)), the sequence of which is includedin the sequence listing as SEQ ID NO: 1. The peptide having the sequenceof SEQ ID NO: 1 may also be designated “native” GLP-1.

In the sequence listing, the first amino acid residue of SEQ ID NO: 1(histidine) is assigned no. 1. However, in what follows—according toestablished practice in the art—this histidine residue is referred to asno. 7, and subsequent amino acid residues are numbered accordingly,ending with glycine no. 37. Therefore, generally, any reference hereinto an amino acid residue number or a position number of the GLP-1(7-37)sequence is to the sequence starting with His at position 7 and endingwith Gly at position 37.

GLP-1 analogues of the derivatives of the invention may be described byreference to i) the number of the amino acid residue in nativeGLP-1(7-37) which corresponds to the amino acid residue which ismodified (i.e., the corresponding position in native GLP-1), and to ii)the actual modification. The following are non-limiting examples ofsuitable analogue nomenclature.

A non-limiting example of a GLP-1 analogue of the derivative of theinvention is an analogue that only is modified so as to comprise a firstlysine residue at a position corresponding to position 37 ofGLP-1(7-37). The amino acid sequence of this analogue is otherwiseidentical to that of native GLP-1, and this analogue may be designatedK³⁷-GLP-1(7-37). This designation represents the amino acid sequence ofnative GLP-1 where glycine at position 37 has been substituted withlysine.

This GLP-1 analogue of the derivative of the invention furthermorecomprises a second lysine residue at a position corresponding toposition 26 of GLP-1(7-37). As the amino acid sequence of this analogueis otherwise identical to that of native GLP-1, such analogue is, still,designated K³⁷-GLP-1(7-37), as K²⁶ is implied by the reference to nativeGLP-1(7-37), SEQ ID NO: 1.

Accordingly, K³⁷-GLP-1(7-37) designates a GLP-1(7-37) analogue whereinthe naturally occurring glycine at position 37 has been substituted withlysine.

The term “analogue of K³⁷-GLP-1(7-37)” refers to an analogue ofGLP-1(7-37) which comprises the modification K³⁷ and at least oneadditional modification, as compared to GLP-1(7-37).

The GLP-1 analogue forming part of the derivative of the inventioncomprises a first K residue at a position corresponding to position 37of GLP-1(7-37) (SEQ ID NO: 1), a second K residue at a positioncorresponding to position 26 of GLP-1(7-37), and a maximum of ten aminoacid modifications as compared to GLP-1(7-37), wherein the first Kresidue is designated K³⁷, and the second K residue is designated K²⁶.In other words, it is a modified GLP-1(7-37) peptide in which a numberof amino acid residues have been changed when compared to nativeGLP-1(7-37) (SEQ ID NO: 1). These changes, or modifications, mayrepresent, independently, one or more amino acid substitutions,additions, and/or deletions.

Another non-limiting example of an analogue of a derivative of theinvention is [Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37), which designates aGLP-1(7-37) analogue, in which the alanine at position 8 has beensubstituted with α-aminoisobutyric acid (Aib), the lysine at position 34has been substituted with arginine, and the glycine at position 37 hasbeen substituted with lysine. This analogue may also be designated(8Aib, R34, K37) GLP-1(7-37).

An additional non-limiting example of an analogue of a derivative of theinvention is an analogue “which comprises 34E, 34Q, or 34R” which refersto a GLP-1 analogue which has either a glutamic acid (E), a glutamine(Q), or an arginine (R) at a position corresponding to position 34 ofnative GLP-1 (SEQ ID NO: 1), and which may comprise furthermodifications as compared to SEQ ID NO: 1.

A still further non-limiting example of an analogue of a derivative ofthe invention is the analogue of GLP-1(7-37) (SEQ ID NO: 1) which issimply designated “(8Aib, 31H, 34Q, 37K)”. This designation refers to ananalogue which is identical to SEQ ID NO: 1 except for these foursubstitutions, i.e. an analogue in which the alanine at position 8 hasbeen substituted with α-aminoisobutyric acid (Aib), the tryptophan atposition 31 has been substituted with histidine, the lysine at position34 has been substituted with glutamine, and the glycine at position 37has been substituted with lysine. This analogue does not comprisefurther modifications as compared to SEQ ID NO: 1.

A still further non-limiting example of an analogue of a derivative ofthe invention is an analogue comprising des7 (or Des), which refers toan analogue of GLP-1(7-37) in which the N-terminal amino acid,histidine, has been deleted. This analogue may also be designatedGLP-1(8-37).

Similarly, (des7+des8); (des7, des8); (des7-8); or (Des⁷, Des⁸) inrelation to an analogue of GLP-1(7-37), where the reference toGLP-1(7-37) may be implied, refers to an analogue in which the aminoacids corresponding to the two N-terminal amino acids of native GLP-1,histidine and alanine, have been deleted. This analogue may also bedesignated GLP-1(9-37).

A still further non-limiting example of an analogue of a derivative ofthe invention is an analogue comprising Imp⁷, and/or (Aib⁸ or S⁸), whichrefers to a GLP-1(7-37) analogue, which, when compared to native GLP-1,comprises a substitution of histidine at position 7 withimidazopropionic acid (Imp); and/or a substitution of alanine atposition 8 with α-aminoisobutyric acid (Aib), or with serine.

Analogues “comprising” certain specified modifications may comprisefurther modifications, when compared to SEQ ID NO: 1. Two examples,non-limiting, of analogues comprising Imp⁷, and/or (Aib⁸ or S⁸), andforming part of derivatives of the invention are the peptide parts ofChem. 47 and Chem. 58.

Non-limiting examples of an analogue of GLP-1(7-37) comprising(des7+des8), Arg34, Lys37, and Glu38 are the following: [Des⁷, Des⁸,Arg³⁴, Lys³⁷]GLP-1(7-37)-Glu³⁸ peptide; andN⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}[Arg³⁴,Lys³⁷]GLP-1(9-37)Glu³⁸-peptide. In the latter compound a dipeptidemimetic of the N-terminus of native GLP-1 (His-Ala) is attached to thenew N-terminus, Glu 9, via an amide bond.

Suitable His- or His-Ala mimetics that may be used as a kind of asubstitute for the deleted N-terminal amino acids, if any, comprise aheterocyclic, nitrogen-containing, aromatic ring structure, e.g.pyridine or imidazole. Preferred His- or His-Ala mimetics arederivatives of an imidazole or a pyridine, other than His and His-Ala,in one embodiment having a substituent with a free carboylic acid group,which can form an amide bond with an amino group of the N-terminal aminoacid of the peptide. The term imidazole refers to imidazoles as a classof heterocycles with similar ring structure but varying substituents,and vice-versa for pyridine.

As is apparent from the above examples, amino acid residues may beidentified by their full name, their one-letter code, and/or theirthree-letter code. These three ways are fully equivalent.

The expressions “a position equivalent to” or “corresponding position”may be used to characterise the site of modification in a modifiedGLP-1(7-37) sequence by reference to native GLP-1(7-37) (SEQ ID NO: 1).Equivalent or corresponding positions, as well as the number ofmodifications, are easily deduced, e.g. by simple handwriting andeyeballing; and/or a standard protein or peptide alignment program maybe used, such as “align” which is a Needleman-Wunsch alignment. Thealgorithm is described in Needleman, S. B. and Wunsch, C. D., (1970),Journal of Molecular Biology, 48: 443-453, and the align program byMyers and W. Miller in “Optimal Alignments in Linear Space” CABIOS(computer applications in the biosciences) (1988) 4:11-17. For thealignment, the default scoring matrix BLOSUM50 and the default identitymatrix may be used, and the penalty for the first residue in a gap maybe set at −12, or preferably at −10, and the penalties for additionalresidues in a gap at −2, or preferably at −0.5.

An example of such alignment is inserted hereinbelow, in which sequenceno. 1 is SEQ ID NO: 1, and sequence no. 2 (SEQ ID NO: 2) is the analogue(des7-8, 34R, 37K, 38E) thereof:

In case of non-natural amino acids such as Imp and/or Aib being includedin the sequence, or in case of His-Ala mimetics, these may, foralignment purposes, be replaced with X. If desired, X can later bemanually corrected.

The term “peptide”, as e.g. used in the context of the GLP-1 analoguesof the derivatives of the invention, refers to a compound whichcomprises a series of amino acids intereconnected by amide (or peptide)bonds.

In a particular embodiment the peptide is to a large extent, orpredominantly, composed of amino acids interconnected by amide bonds(e.g., at least 50%, 60%, 70%, 80%, or at least 90%, by molar mass). Inanother particular embodiment the peptide consists of amino acidsinterconnected by peptide bonds.

The peptides of the invention comprise at least five constituent aminoacids connected by peptide bonds. In particular embodiments the peptidecomprises at least 10, preferably at least 15, more preferably at least20, even more preferably at least 25, or most preferably at least 28amino acids.

In particular embodiments, the peptide is composed of at least fiveconstituent amino acids, preferably composed of at least 10, at least15, at least 20, at least 25, or most preferably composed of at least 28amino acids.

In additional particular embodiments, the peptide is a) composed of, orb) consists of, i) 29, ii) 30, iii) 31, or iv) 32 amino acids.

In a still further particular embodiment the peptide consists of aminoacids interconnected by peptide bonds.

Amino acids are molecules containing an amine group and a carboxylicacid group, and, optionally, one or more additional groups, oftenreferred to as a side chain.

The term “amino acid” includes proteogenic amino acids (encoded by thegenetic code, including natural amino acids, and standard amino acids),as well as non-proteogenic (not found in proteins, and/or not coded forin the standard genetic code), and synthetic amino acids. Thus, theamino acids may be selected from the group of proteinogenic amino acids,non-proteinogenic amino acids, and/or synthetic amino acids.

Non-limiting examples of amino acids which are not encoded by thegenetic code are gamma-carboxyglutamate, ornithine, and phosphoserine.Non-limiting examples of synthetic amino acids are the D-isomers of theamino acids such as D-alanine (in what follows sometimes abbreviated “a”as f.ex. in “a8”, which accordingly refers to D-Ala⁸) and D-leucine, Aib(α-aminoisobutyric acid), β-alanine, and des-amino-histidine (desH,alternative name imidazopropionic acid, abbreviated Imp).

In what follows, all amino acids for which the optical isomer is notstated is to be understood to mean the L-isomer (unless otherwisespecified).

The GLP-1 derivatives and analogues of the invention have GLP-1activity. This term refers to the ability to bind to the GLP-1 receptorand initiate a signal transduction pathway resulting in insulinotropicaction or other physiological effects as is known in the art. Forexample, the analogues and derivatives of the invention can be testedfor GLP-1 activity using the assay described in Example 50 herein.

GLP-1 Derivatives

The term “derivative” as used herein in the context of a GLP-1 peptideor analogue means a chemically modified GLP-1 peptide or analogue, inwhich one or more substituents have been covalently attached to thepeptide. The substituent may also be referred to as a side chain.

In a particular embodiment, the side chain is capable of formingnon-covalent aggregates with albumin, thereby promoting the circulationof the derivative with the blood stream, and also having the effect ofprotracting the time of action of the derivative, due to the fact thatthe aggregate of the GLP-1-derivative and albumin is only slowlydisintegrated to release the active pharmaceutical ingredient. Thus, thesubstituent, or side chain, as a whole is preferably referred to as analbumin binding moiety.

In particular embodiments, the side chain has at least 10 carbon atoms,or at least 15, 20, 25, 30, 35, or at least 40 carbon atoms. In furtherparticular embodiments, the side chain may further include at least 5hetero atoms, in particular O and N, for example at least 7, 9, 10, 12,15, 17, or at least 20 hetero atoms, such as at least 1, 2, or 3N-atoms, and/or at least 3, 6, 9, 12, or 15 O-atoms.

In another particular embodiment the albumin binding moiety comprises aportion which is particularly relevant for the albumin binding andthereby the protraction, which portion may accordingly be referred to asa protracting moiety. The protracting moiety may be at, or near, theopposite end of the albumin binding moiety, relative to its point ofattachment to the peptide.

In a still further particular embodiment the albumin binding moietycomprises a portion inbetween the protracting moiety and the point ofattachment to the peptide, which portion may be referred to as a linker,linker moiety, spacer, or the like. The linker may be optional, andhence in that case the albumin binding moiety may be identical to theprotracting moiety.

In particular embodiments, the albumin binding moiety and/or theprotracting moiety is lipophilic, and/or negatively charged atphysiological pH (7.4).

The albumin binding moiety, the protracting moiety, or the linker may becovalently attached to a lysine residue of the GLP-1 peptide byacylation. Additional or alternative conjugation chemistry includesalkylation, ester formation, or amide formation, or coupling to acysteine residue, such as by maleimide or haloacetamide (such asbromo-/fluoro-/iodo-) coupling.

In a preferred embodiment, an active ester of the albumin bindingmoiety, preferably comprising a protracting moiety and a linker, iscovalently linked to an amino group of a lysine residue, preferably theepsilon amino group thereof, under formation of an amide bond (thisprocess being referred to as acylation).

Unless otherwise stated, when reference is made to an acylation of alysine residue, it is understood to be to the epsilon-amino groupthereof.

A derivative comprising two protracting moieties attached to K²⁶ andK³⁷, optionally via a linker, may be referred to as a derivative whichhas been acylated twice, double-acylated, or dual acylated at theepsilon-amino groups of the lysine residues at positions correspondingto position 26 and 37, respectively, of GLP-1(7-37).

For the present purposes, the terms “albumin binding moiety”,“protracting moiety”, and “linker” may include the unreacted as well asthe reacted forms of these molecules. Whether or not one or the otherform is meant is clear from the context in which the term is used.

In one aspect, each protracting moiety comprises, or consists of, aprotracting moiety independently selected from Chem. 1, Chem. 2, Chem.3, and Chem. 4:HOOC—(CH₂)_(x)—CO—*  Chem. 1:HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:R¹—C₆H₄—(CH₂)_(z)—CO—*  Chem. 3:HOOC—C₄SH₂—(CH₂)_(w)—CO—*  Chem. 4:

in which x is an integer in the range of 6-18, y is an integer in therange of 3-17, z is an integer in the range of 1-5, R¹ is a group havinga molar mass not higher than 150 Da, and w is an integer in the range of6-18.

In one embodiment, *—(CH₂)_(x)—* refers to straight or branched,preferably straight, alkylene in which x is an integer in the range of6-18.

In another embodiment, *—(CH₂)_(y)—* refers to straight or branched,preferably straight, alkylene in which y is an integer in the range of3-17.

In a third embodiment, *—(CH₂)_(z)—* refers to straight or branched,preferably straight, alkylene in which z is an integer in the range of1-5.

In a still further embodiment, *—(CH₂)_(w)—* refers to straight orbranched, preferably straight, alkylene in which w is an integer in therange of 6-18.

In another aspect the albumin binding moiety comprises, or consists of,a protracting moiety selected from fatty diacids, and fatty acids with adistal (terminal) phenyl or phenoxy group, both optionally substituted.Optional substituents to the phenyl, and/or the phenoxy group, have amolar mass not higher than 150 Da, preferably not higher than 125 Da,more preferably not higher than 100 Da, even more preferably not higherthan 75 Da, or most preferably not higher than 50 Da. Examples ofsubstituents include, without limitation, carboxy, hydroxyl, lowerlinear or branched C1-C5 alkyl such as methyl and tert. butyl, andhalogen such as iodine.

For the attachment to the GLP-1 peptide, the acid group of the fattyacid, or one of the acid groups of the fatty diacid, forms an amide bondwith the epsilon amino group of a lysine residue in the GLP-1 peptide,preferably via a linker.

The term “fatty acid” refers to aliphatic monocarboxylic acids havingfrom 4 to 28 carbon atoms, it is preferably unbranched, and/or evennumbered, and it may be saturated or unsaturated.

The term “fatty diacid” refers to fatty acids as defined above but withan additional carboxylic acid group in the omega position. Thus, fattydiacids are dicarboxylic acids.

In a preferred embodiment the protracting moiety is selected fromHOOC—(CH₂)_(n)—CO—*, HOOC—C₆H₄—O—(CH₂)_(m)—CO—*, andR¹—C₆H₄—(CH₂)_(p)—CO—*, in which n is an integer in the range of 8-16, mis an integer in the range of 7-17, p is an integer in the range of 1-5,and R¹ is a group having a molar mass not higher than 150 Da.

The nomenclature is as is usual in the art, for example in the aboveformulas *—COOH as well as HOOC—* refers to carboxy; *—C₆H₄—* tophenylene; *—CO—*, as well as *—OC—*, to carbonyl (O═C<**); C₆H₅—O—* tophenoxy; C₄H₄ S or C₄H₄ to thiophene; and *—C₄SH₂* to a di-radicalthereof (any thiophenylene). In particular embodiments, the aromatics,such as the phenoxy, and the phenylene radicals, may be, independently,ortho, meta, or para. In another embodiment, the thiophenylenedi-radical may be 2,3-; 2,4-; or 2,5-.

The molar mass (M) of a chemical substance (such as the group R¹) is themass of one mole of the substance. The molar mass is quoted in dalton,symbol Da, with the definition 1 Da=1 g/mol.

Molar mass may be calculated from standard atomic weights, and is oftenlisted in chemical catalogues. The molar mass of a compound is given bythe sum of the standard atomic weights of the atoms which form thecompound multiplied by the molar mass constant, M_(u) which equals 1g/mol. As an example, the molecular mass of tert. butyl (C₄H₉) isM(C₄H₉)=([4×12.01]+[9×1.008])×1 g/mol=57 Da.

Standard atomic weights are published by the International Union of Pureand Applied Chemistry (IUPAC), and also reprinted in a wide variety oftextbooks, commercial catalogues, wallcharts etc.

As explained above, the GLP-1 derivatives of the present invention aredouble-acylated, i.e. two albumin binding moieties are covalentlyattached to the GLP-1 peptide. The points of attachment are the nativelysine residue at the position corresponding to position 26 ofGLP-1(7-37), and a lysine residue which has been substituted for thenative glycine residue at the position corresponding to position 37 ofGLP-1(7-37).

In a particular embodiment, the two albumin binding moieites (i.e. theentire side chains) are similar, preferably substantially identical, or,most preferably, identical.

In another particular embodiment, the two protracting moieties aresimilar, preferably substantially identical, or, most preferably,identical.

In a still further particular embodiment, the two linkers are similar,preferably substantially identical, or, most preferably identical.

The term “substantially identical” includes differences from identitywhich are due to formation of one or more salts, esters, and/or amides;preferably formation of one or more salts, methyl esters, and simpleamides; more preferably formation of no more than two salts, methylesters, and/or simple amides; even more preferably formation of no morethan one salt, methyl ester, and/or simple amide; or most preferablyformation of no more than one salt.

In the context of chemical compounds such as the albumin bindingmoieities, protracting moieties, and linkers, similarity and/or identitymay be determined using any suitable computer program and/or algorithmknown in the art.

For example, the similarity of two protracting moieties, two linkers,and/or two entire side chains may suitably be determined using molecularfingerprints. Fingerprints is a mathematical method of representing achemical structure (see e.g. Chemoinformatics: A textbook, JohannGasteiger and Thomas Engel (Eds), Wiley-VCH Verlag, 2003).

Examples of suitable fingerprints include, without limitation, UNITYfingerprints, MDL fingerprints, and/or ECFP fingerprints, such asECFP_(—)6 fingerprints (ECFP stands for extended-connectivityfingerprints).

In particular embodiments, the two protracting moieties, the twolinkers, and/or the two entire side chains are represented as a)ECFP_(—)6 fingerprints; b) UNITY fingerprints; and/or c) MDLfingerprints.

The Tanimoto coefficient is preferably used for calculating thesimilarity of the two fingerprints, whether a), b) or c) is used.

In particular embodiments, whether a), b) or c) is used, the twoprotracting moieties, the two linkers, and/or the two entire sidechains, respectively, have a similarity of at least 0.5 (50%);preferably at least 0.6 (60%); more preferably at least 0.7 (70%), or atleast 0.8 (80%); even more preferably at least 0.9 (90%); or mostpreferably at least 0.99 (99%), such as a similarity of 1.0 (100%).

UNITY fingerprints may be calculated using the programme SYBYL(available from Tripos, 1699 South Hanley Road, St. Louis, Mo.63144-2319 USA). ECFP_(—)6 and MDL fingerprints may be calculated usingthe programme Pipeline Pilot (available from Accelrys Inc., 10188Telesis Court, Suite 100, San Diego, Calif. 92121, USA).

For more details, see for example J. Chem. Inf. Model. 2008, 48,542-549; J. Chem. Inf. Comput. Sci. 2004, 44, 170-178; J. Med. Chem.2004, 47, 2743-2749; J. Chem. Inf. Model. 2010, 50, 742-754; as well asSciTegic Pipeline Pilot Chemistry Collection: Basic Chemistry UserGuide, March 2008, SciTegic Pipeline Pilot Data Modeling Collection,2008-both from Accelrys Software Inc., San Diego, US, and the guideswww.tripos.com/tripos_resources/fileroot/pdfs/Unity_(—)111408.pdf, andwww.tripos.com/data/SYBYL/SYBYL_(—)072505.pdf.

An example of a similarity calculation is inserted hereinbelow, in whichthe entire side chain of Chem. 23 was compared with a methyl esterthereof, viz. the mono methyl ester of the glutamine linker moiety (Chem23a):

Using a) ECFP_(—)6 fingerprints the similarity is 0.798, using b) UNITYfingerprints the similarity is 0.957; and using MDL fingerprints thesimilarity is 0.905.

In case of two identical side chains (albumin binding moieties) thederivative may be designated symmetrical.

In particular embodiments, the similarity coefficient is at least 0.80,preferably at least 0.85, more preferably at least 0.90, even morepreferably at least 0.95, or most preferably at least 0.99.

Each of the two linkers of the derivative of the invention may comprisethe following first linker element:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5.

In a particular embodiment, when k=1 and n=1, this linker element may bedesignated OEG, or a di-radical of 8-amino-3,6-dioxaoctanic acid, and/orit may be represented by the following formula:*—NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—CO—*.  Chem. 5a:

In another particular embodiment, each linker of the derivative of theinvention may further comprise, independently, a second linker element,preferably a Glu di-radical, such as Chem. 6 and/or Chem. 7:

wherein the Glu di-radical may be included p times, where p is aninteger in the range of 1-3.

Chem. 6 may also be referred to as gamma-Glu, or briefly gGlu, due tothe fact that it is the gamma carboxy group of the amino acid glutamicacid which is here used for connection to another linker element, or tothe epsilon-amino group of lysine. As explained above, the other linkerelement may, for example, be another Glu residue, or an OEG molecule.The amino group of Glu in turn forms an amide bond with the carboxygroup of the protracting moiety, or with the carboxy group of, e.g., anOEG molecule, if present, or with the gamma-carboxy group of, e.g.,another Glu, if present.

Chem. 7 may also be referred to as alpha-Glu, or briefly aGlu, or simplyGlu, due to the fact that it is the alpha carboxy group of the aminoacid glutamic acid which is here used for connection to another linkerelement, or to the epsilon-amino group of lysine.

The above structures of Chem. 6 and Chem. 7 cover the L-form, as well asthe D-form of Glu. In particular embodiments, Chem. 6 and/or Chem. 7is/are, independently, a) in the L-form, or b) in the D-form.

In another particular embodiment, each linker of the derivative of theinvention may further comprise, independently, the following thirdlinker element:*—NH—(CH₂)_(q)—CHR²—CO—*,  Chem. 8:in which q is an integer in the range of 2-12, and R² is hydrogen (H) oramino (NH₂).

In Chem. 8, the group *—(CH₂)_(q)—* may represent straight or branched,preferably straight, alkylene, wherein q is an integer in the range of2-12.

In still further particular embodiments the linker has a) from 5 to 41C-atoms; and/or b) from 4 to 28 hetero atoms. Particular andnon-limiting examples of hetero atoms are N-, and O-atoms. H-atoms arenot hetero atoms.

Alternatively, the linker moiety, if present, has from 5 to 30 C-atoms,preferably from 5 to 25 C-atoms, more preferably from 5 to 20 C-atoms,or most preferably from 5 to 17 C-atoms. In additional preferredembodiments, the linker moiety, if present, has from 4 to 20 heteroatoms, preferably from 4 to 18 hetero atoms, more preferably from 4 to14 hetero atoms, or most preferably from 4 to 12 hetero atoms.

Alternatively, the linker comprises at least one OEG molecule, and/or atleast one glutamic acid residue, or rather the corresponding radicals.

In a particular embodiment, each linker consists of one time Chem. 6 andtwo times Chem. 5, interconnected via amide bonds and in the sequenceindicated, the linker being connected at its free amino end to the freecarbonyl group of the protracting moiety, and at its free carbonyl endto the epsilon amino group of K²⁶ or K³⁷ of the GLP-1 analogue.

In another particular embodiment, each linker consists of two timesChem. 5 and one time Chem. 6, interconnected via amide bonds and in thesequence indicated, the linker being connected at its free amino end tothe free carbonyl group of the protracting moiety, and at its freecarbonyl end to the epsilon amino group of K²⁶ or K³⁷ of the GLP-1analogue.

The derivatives of the invention may exist in different stereoisomericforms having the same molecular formula and sequence of bonded atoms,but differing only in the three-dimensional orientation of their atomsin space. The stereoisomerism of the examplified derivatives of theinvention is indicated in the experimental section, in the names as wellas the structures, using standard nomenclature. Unless otherwise statedthe invention relates to all stereoisomeric forms of the claimedderivative.

The concentration in plasma of the GLP-1 derivatives of the inventionmay be determined using any suitable method. For example, LC-MS (LiquidChromatography Mass Spectroscopy) may be used, or immunoassays such asRIA (Radio Immuno Assay), ELISA (Enzyme-Linked Immuno Sorbent Assay),and LOCI (Luminescence Oxygen Channeling Immunoasssay). Generalprotocols for suitable RIA and ELISA assays are found in, e.g.,WO09/030,738 on p. 116-118. A preferred assay is the LOCI assaydescribed in Example 52, 55, and 58 herein.

Pharmaceutically Acceptable Salt, Amide, or Ester

The derivatives, analogues, and intermediate products of the inventionmay be in the form of a pharmaceutically acceptable salt, amide, orester.

Salts are e.g. formed by a chemical reaction between a base and an acid,e.g.: 2NH₃+H₂SO₄→(NH₄)₂SO₄.

The salt may be a basic salt, an acid salt, or it may be neither nor(i.e. a neutral salt). Basic salts produce hydroxide ions and acid saltshydronium ions in water.

The salts of the derivatives of the invention may be formed with addedcations or anions that react with anionic or cationic groups,respectively. These groups may be situated in the peptide moiety, and/orin the side chain of the derivatives of the invention.

Non-limiting examples of anionic groups of the derivatives of theinvention include free carboxylic groups in the side chain, if any, aswell as in the peptide moiety. The peptide moiety often includes a freecarboxylic acid group at the C-terminus, and it may also include freecarboxylic groups at internal acid amino acid residues such as Asp andGlu.

Non-limiting examples of cationic groups in the peptide moiety includethe free amino group at the N-terminus, if present, as well as any freeamino group of internal basic amino acid residues such as His, Arg, andLys.

The ester of the derivatives of the invention may, e.g., be formed bythe reaction of a free carboxylic acid group with an alcohol or aphenol, which leads to replacement of at least one hydroxyl group by analkoxy or aryloxy group

The ester formation may involve the free carboxylic group at theC-terminus of the peptide, and/or any free carboxylic group in the sidechain.

The amide of the derivatives of the invention may, e.g., be formed bythe reaction of a free carboxylic acid group with an amine or asubstituted amine, or by reaction of a free or substituted amino groupwith a carboxylic acid.

The amide formation may involve the free carboxylic group at theC-terminus of the peptide, any free carboxylic group in the side chain,the free amino group at the N-terminus of the peptide, and/or any freeor substituted amino group of the peptide in the peptide and/or the sidechain.

In a particular embodiment, the peptide or derivative is in the form ofa pharmaceutically acceptable salt. In another particular embodiment,the derivative is in the form of a pharmaceutically acceptable amide,preferably with an amide group at the C-terminus of the peptide. In astill further particular embodiment, the peptide or derivative is in theform a pharmaceutically acceptable ester.

Intermediate Products

One type of intermediate product of the invention takes the form of aGLP-1 analogue which comprises the following modifications as comparedto GLP-1(7-37) (SEQ ID NO: 1): (i) (8Aib, 31H, 34Q, 37K); (ii) (des7-8,34R, 37K, 38E); (iii) (des7-8, 34R, 37K); (iv) (8Aib, 9G, 34R, 37K); (v)(8Aib, 23R, 34R, 37K); (vi) (31H, 34Q, 37K); (vii) (9Q, 34R, 37K); (iix)(30E, 34R, 37K); (ix) (34R, 37K, 38G); (x) (34R, 36G, 37K); or (xi)(34R, 37K, 38E); or a pharmaceutically acceptable, salt, amide, or esterthereof.

Another type of intermediate product of the invention takes the form ofan albumin binding moiety, or a side chain intermediate, comprising aprotracting moiety selected from Chem. 2c, Chem. 3b, and Chem. 4b:HOOC—C₆H₄—O—(CH₂)_(y)—CO—PG  Chem. 2c:R¹—C₆H₄—(CH₂)_(z)—CO—PG  Chem. 3b:HOOC—C₄SH₂—(CH₂)_(w)—CO—PG  Chem. 4b:

in which y is an integer in the range of 3-17, z is an integer in therange of 1-5, R¹ is a group having a molar mass not higher than 150 Da,w is an integer in the range of 6-18, whee PG is a protection group,preferably *—CO—PG is an activated ester; wherein, optionally, the other(distal) *—COOH group of the protracting moiety, if present, ispreferably also protected as is known in the art, for examplefunctionalised as a non-reactive ester; or a pharmaceutically acceptablesalt, amide, or ester thereof.

In a particular embodiment, the side chain intermediate comprises

a) a protracting moiety selected from Chem. 2, Chem. 3, and Chem. 4:HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:R¹—C₆H₄—(CH₂)_(z)—CO—*  Chem. 3:HOOC—C₄SH₂—(CH₂)_(w)—CO—*  Chem. 4:in which y is an integer in the range of 3-17, z is an integer in therange of 1-5, R¹ is a group having a molar mass not higher than 150 Da,and w is an integer in the range of 6-18; and b) one or more linkersselected from Chem. 5b, Chem. 6, and Chem. 7:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5; and PG is a protection group; wherein, optionally, the*—COOH group of the protracting moiety is preferably also protected asis known in the art, preferably functionalised as a non-reactive ester;or a pharmaceutically acceptable salt, amide, or ester thereof.

In a particular embodiment, PG is a group that reversibly renders thecompound such as the protracting moiety unreactive, and that can beremoved selectively.

Non-limiting examples of PG groups are —OH, or groups functionalised asan activated ester, for example, without limitation, OPfp, OPnp, andOSuc.

Other suitable activated esters may be selected, e.g., according to theteaching of M. Bodanszky, “Principles of Peptide Synthesis”, 2nd ed.,Springer Verlag, 1993.

Functional Properties

In a first functional aspect, the derivatives of the invention have agood potency. Also, or alternatively, in a second functional aspect,they have a protracted pharmacokinetic profile. Also, or alternatively,in a third functional aspect, they are stable against degradation bygastro intestinal enzymes. Also, or alternatively, in a fourthfunctional aspect, they have a high oral bioavailability.

Biological Activity (Potency)

According to the first functional aspect, the derivatives of theinvention, as well as the constituent GLP-1 peptides as such (such asK³⁷-GLP-1(7-37) or analogues thereof), are biologically active, orpotent.

In a particular embodiment, potency and/or activity refers to in vitropotency, i.e. performance in a functional GLP-1 receptor assay, more inparticular to the capability of stimulating cAMP formation in a cellline expressing the cloned human GLP-1 receptor.

The stimulation of the formation of cAMP in a medium containing thehuman GLP-1 receptor may preferably be determined using a stabletransfected cell-line such as BHK467-12A (tk-ts13), and/or using for thedetermination of cAMP a functional receptor assay, e.g. based oncompetition between endogenously formed cAMP and exogenously addedbiotin-labelled cAMP, in which assay cAMP is more preferably capturedusing a specific antibody, and/or wherein an even more preferred assayis the AlphaScreen cAMP Assay, most preferably the one described inExample 50.

The term half maximal effective concentration (EC_(K)) generally refersto the concentration which induces a response halfway between thebaseline and maximum, by reference to the dose response curve. EC₅₀ isused as a measure of the potency of a compound and represents theconcentration where 50% of its maximal effect is observed.

The in vitro potency of the derivatives of the invention may bedetermined as described above, and the EC₅₀ of the derivative inquestion determined. The lower the EC₅₀, the better the potency.

In a particular embodiment, the medium has the following composition(final in-assay concentrations): 50 mM TRIS-HCl; 5 mM HEPES; 10 mMMgCl₂, 6H₂O; 150 mM NaCl; 0.01% Tween™ 0.1% BSA ; 0.5 mM IBMX; 1 mM ATP;1 uM GTP; pH 7.4.

An alternative medium is: 50 mM Tris-HCl, 1 mM EGTA, 1.5 mM MgSO₄, 1.7mM ATP, 20 mM GTP, 2 mM 3-isobutyl-1-methylxanthine (IBMX), 0.01%Tween-20, pH 7.4.

In a further particular embodiment, the derivative of the invention hasan EC₅₀ at or below 3000 μM, more preferably below 2000 μM, even morepreferably below 1000 μM, or most preferably below 500 μM.

In another particular embodiment the derivatives of the invention arepotent in vivo, which may be determined as is known in the art in anysuitable animal model, as well as in clinical trials.

The diabetic db/db mouse is one example of a suitable animal model, andthe blood glucose lowering effect may be determined in such mice invivo, e.g. as described in Example 53, or as described in Example 43 ofWO09/030,738.

Also, or alternatively, the effect on glucose mediated insulin secretionin vivo may be determined in pharmacodynamic studies in minipigs(IVGTT), e.g. as described in Example 55.

Also, or alternatively, the effect on feed intake in vivo may bedetermined in pharmacodynamic studies in pigs, e.g. as described inExample 56.

Protraction—Receptor Binding/Low and High Albumin

According to the second functional aspect, the derivatives of theinvention are protracted.

The ability of the derivatives of the invention to bind to the GLP-1receptor in the presence of a low and a high concentration of albumin,respectively, may be determined as described in Example 51.

Generally, the binding to the GLP-1 receptor at low albuminconcentration should be as good as possible, corresponding to a low IC₅₀value.

The IC₅₀ value at high albumin concentration is a measure of theinfluence of albumin on the binding of the derivative to the GLP-1receptor. As is known, the GLP-1 derivatives also bind to albumin. Thisis a generally desirable effect, which extends their lifetime in plasma.Therefore, the IC₅₀ value at high albumin will generally be higher thanthe IC₅₀ value at low albumin, corresponding to a reduced binding to theGLP-1 receptor, caused by albumin binding competing with the binding tothe GLP-1 receptor.

A high ratio (IC₅₀ value (high albumin)/IC₅₀ value (low albumin)) maytherefore be taken as an indication that the derivative in questionbinds well to albumin (may have a long half-life), and also per se bindswell to the GLP-1 receptor (the IC₅₀ value (high albumin) is high, andthe IC₅₀ value (low albumin) is low). On the other hand, albumin bindingmay not always be desirable, or the binding to albumin may become toostrong. Therefore, the desirable ranges for IC₅₀ (low albumin), IC₅₀(high albumin)/, and the ratio high/low may vary from compound tocompound, depending on the intended use and the circumstancessurrounding such use, and on other compound properties of potentialinterest.

In a particular embodiment, the GLP-1 receptor binding affinity (IC₅₀)in the presence of 0.005% HSA (low albumin) is below 1000.00 nM,preferably below 600.00 nM, more preferably below 100.00 nM, or mostpreferably below 50.00 nM.

A suitable assay for determining receptor binding at high and lowalbumin concentration is disclosed in Example 51 herein.

Protraction—Half Life In Vivo in Rats

According to the second functional aspect, the derivatives of theinvention are protracted. In a particular embodiment, protraction may bedetermined as half-life (T_(1/2)) in vivo in rats after i.v.administration. In additional embodiments, the half-life is at least 4hours, preferably at least 6 hours, even more preferably at least 8hours, or most preferably at least 10 hours.

A suitable assay for determining half-life in vivo in rats after i.v.administration is disclosed in Example 58 herein.

Protraction—Half Life In Vivo in Minipigs

According to the second functional aspect, the derivatives of theinvention are protracted. In a particular embodiment protraction may bedetermined as half-life (T_(1/2)) in vivo in minipigs after i.v.administration. In additional embodiments, the half-life is at least 12hours, preferably at least 24 hours, more preferably at least 36 hours,even more preferably at least 48 hours, or most preferably at least 60hours.

A suitable assay for determining half-life in vivo in minipigs afteri.v. administration is disclosed in Example 54 herein.

Degradation by Gastro Intestinal Enzymes

According to the third functional aspect, the derivatives of theinvention are stable, or stabilised, against degradation by one or moregastro intestinal enzymes.

Gastro intestinal enzymes include, without limitation, exo and endopeptidases, such as pepsin, trypsin, chymotrypsin, elastases, andcarboxypeptidases. The stability may be tested against these gastrointestinal enzymes in the form of purified enzymes, or in the form ofextracts from the gastrointestinal system.

In a particular embodiment, the derivative of the invention has an invitro half-life (T_(1/2)), in an extract of rat small intestines,divided by the corresponding half-life (T_(1/2)) of GLP-1(7-37), of atleast 1, preferably above 1.0, more preferably at least 1.2, still morepreferably at least 2.0, even more preferably at least 3.0, or mostpreferably at least 4.0. In other words, a ratio(SI) may be defined foreach derivative, viz. as the in vitro half-life (T_(1/2)) of thederivative in question, in an extract of rat small intestines, dividedby the corresponding half-life (T_(1/2)) of GLP-1(7-37).

A suitable assay for determining in vitro half-life in an extract of ratsmall intestines is disclosed in Example 57 herein.

Oral Bioavailability

According to the fourth functional aspect, the derivatives of theinvention have a high oral bioavailability.

The oral bioavailability of commercial GLP-1 derivatives is very low.The oral bioavailability of GLP-1 derivatives under development for i.v.or s.c. administration is also low.

Accordingly, there is a need in the art for GLP-1 derivatives of animproved oral bioavailability. Such derivatives could be suitablecandidates for oral administration, as long as their potency isgenerally satisfactory, and/or as long as their half-life is alsogenerally satisfactory.

The present inventors identified a novel class of GLP-1 derivatives,which have a surprisingly high oral bioavailability, and at the sametime a satisfactory potency, and/or half-life.

Also, or alternatively, these derivatives have a surprisingly high oralbioavailability, and at the same time a high binding affinity (i.e. alow IC₅₀ value) to the GLP-1 receptor at a low concentration of albumin.

These features are of importance with a view to obtaining a low dailyoral dose of the active pharmaceutical ingredient, which is desirablefor various reasons, including, e.g., economy of production, likelihoodof potential safety issues, as well as administration comfort issues,and environmental concerns.

Generally, the term bioavailability refers to the fraction of anadministered dose of the active pharmaceutical ingredient (API), such asa derivative of the invention that reaches the systemic circulationunchanged. By definition, when an API is administered intravenously, itsbioavailability is 100%. However, when it is administered via otherroutes (such as orally), its bioavailability decreases (due toincomplete absorption and first-pass metabolism). Knowledge aboutbioavailability is essential when calculating dosages fornon-intravenous routes of administration.

Absolute oral bioavailability compares the bioavailability (estimated asthe area under the curve, or AUC) of the API in systemic circulationfollowing oral administration, with the bioavailability of the same APIfollowing intravenous administration. It is the fraction of the APIabsorbed through non-intravenous administration compared with thecorresponding intravenous administration of the same API. The comparisonmust be dose normalised if different doses are used; consequently, eachAUC is corrected by dividing the corresponding dose administered.

A plasma API concentration vs time plot is made after both oral andintravenous administration. The absolute bioavailability (F) is thedose-corrected AUC-oral divided by AUC-intravenous.

The derivatives of the invention have an absolute oral bioavailabilitywhich is higher than that of a) liraglutide, and/or b) semaglutide;preferably at least 10% higher, more preferably at least 20% higher,even more preferably at least 30% higher, or most preferably at least40% higher. Before testing oral bioavailability the derivatives of theinvention may suitably be formulated as is known in the art of oralformulations of insulinotropic compounds, e.g. using any one or more ofthe formulations described in WO 2008/145728.

A test has been developed, described in Example 52, which was found tobe a very good prediction of oral bioavailability. According to thistest, after direct injection of the GLP-1 derivative into the intestinallumen of rats, the concentration (exposure) thereof in plasma isdetermined, and the ratio of plasma concentration (pmol/l) divided bythe concentration of the dosing solution (umol/l) is calculated for t=30min. This ratio is a measure of intestinal bioavailability, and it hasshown to correlate nicely with actual oral bioavailability data.

Additional particular embodiments of the derivatives of the inventionare described in the sections headed “particular embodiments” and“additional particular embodiments” before the experimental section.

Production Processes

The production of peptides like GLP-1(7-37) and GLP-1 analogues is wellknown in the art.

The GLP-1 moiety of the derivatives of the invention (or fragmentsthereof), such as K³⁷-GLP-1(7-37) or an analogue or fragment thereof,may for instance be produced by classical peptide synthesis, e.g., solidphase peptide synthesis using t-Boc or Fmoc chemistry or other wellestablished techniques, see, e.g., Greene and Wuts, “Protective Groupsin Organic Synthesis”, John Wiley & Sons, 1999, Florencio ZaragozaDorwald, “Organic Synthesis on solid Phase”, Wiley-VCH Verlag GmbH,2000, and “Fmoc Solid Phase Peptide Synthesis”, Edited by W. C. Chan andP. D. White, Oxford University Press, 2000.

Also, or alternatively, they may be produced by recombinant methods,viz. by culturing a host cell containing a DNA sequence encoding theanalogue and capable of expressing the peptide in a suitable nutrientmedium under conditions permitting the expression of the peptide.Non-limiting examples of host cells suitable for expression of thesepeptides are: Escherichia coli, Saccharomyces cerevisiae, as well asmammalian BHK or CHO cell lines.

Those derivatives of the invention which include non-natural amino acidsand/or a covalently attached N-terminal mono- or dipeptide mimetic maye.g. be produced as described in the experimental part. Or see e.g.,Hodgson et al: “The synthesis of peptides and proteins containingnon-natural amino acids”, Chemical Society Reviews, vol. 33, no. 7(2004), p. 422-430; and WO 2009/083549 A1 entitled “Semi-recombinantpreparation of GLP-1 analogues”.

Specific examples of methods of preparing a number of the derivatives ofthe invention are included in the experimental part.

Pharmaceutical Compositions

Pharmaceutical compositions comprising a derivative of the invention ora pharmaceutically acceptable salt, amide, or ester thereof, and apharmaceutically acceptable excipient may be prepared as is known in theart.

The term “excipient” broadly refers to any component other than theactive therapeutic ingredient(s). The excipient may be an inertsubstance, an inactive substance, and/or a not medicinally activesubstance.

The excipient may serve various purposes, e.g. as a carrier, vehicle,diluent, tablet aid, and/or to improve administration, and/or absorptionof the active substance.

The formulation of pharmaceutically active ingredients with variousexcipients is known in the art, see e.g. Remington: The Science andPractice of Pharmacy (e.g. 19^(th) edition (1995), and any latereditions).

Non-limiting examples of excipients are: Solvents, diluents, buffers,preservatives, tonicity regulating agents, chelating agents, andstabilisers.

Examples of formulations include liquid formulations, i.e. aqueousformulations comprising water. A liquid formulation may be a solution,or a suspension. An aqueous formulation typically comprises at least 50%w/w water, or at least 60%, 70%, 80%, or even at least 90% w/w of water.

Alternatively, a pharmaceutical composition may be a solid formulation,e.g. a freeze-dried or spray-dried composition, which may be used as is,or whereto the physician or the patient adds solvents, and/or diluentsprior to use.

The pH in an aqueous formulation may be anything between pH 3 and pH 10,for example from about 7.0 to about 9.5; or from about 3.0 to about 7.0.

A pharmaceutical composition may comprise a buffer. The buffer may e.g.be selected from the group consisting of sodium acetate, sodiumcarbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine,sodium dihydrogen phosphate, disodium hydrogen phosphate, sodiumphosphate, and tris(hydroxymethyl)-aminomethane, bicine, tricine, malicacid, succinate, maleic acid, fumaric acid, tartaric acid, asparticacid, and mixtures thereof. A pharmaceutical composition may comprise apreservative. The preservative may e.g. be selected from the groupconsisting of phenol, o-cresol, m-cresol, p-cresol, methylp-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butylp-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, andthiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodiumdehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethoniumchloride, chlorphenesine (3p-chlorphenoxypropane-1,2-diol), and mixturesthereof. The preservative may be present in a concentration from 0.1mg/ml to 20 mg/ml. A pharmaceutical composition may comprise an isotonicagent. The isotonic agent may e.g. be selected from the group consistingof a salt (e.g. sodium chloride), a sugar or sugar alcohol, an aminoacid (e.g. glycine, histidine, arginine, lysine, isoleucine, asparticacid, tryptophan, threonine), an alditol (e.g. glycerol (glycerine),1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG400), and mixtures thereof. Any sugar suchas mono-, di-, or polysaccharides, or water-soluble glucans, includingfor example fructose, glucose, mannose, sorbose, xylose, maltose,lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin,alfa and beta HPCD, soluble starch, hydroxyethyl starch andcarboxymethylcellulose—Na may be used. Sugar alcohol is defined as aC4-C8 hydrocarbon having at least one —OH group and includes, forexample, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol,and arabitol. In one embodiment, the sugar alcohol additive is mannitol.A pharmaceutical composition may comprise a chelating agent. Thechelating agent may e.g. be selected from salts ofethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid,and mixtures thereof. A pharmaceutical composition may comprise astabiliser. The stabiliser may e.g. be one or more oxidation inhibitors,aggregation inhibitors, surfactants, and/or one or more proteaseinhibitors. Non-limiting examples of these various kinds of stabilisersare disclosed in the following.

The term “aggregate formation” refers to a physical interaction betweenthe polypeptide molecules resulting in formation of oligomers, which mayremain soluble, or large visible aggregates that precipitate from thesolution. Aggregate formation by a polypeptide during storage of aliquid pharmaceutical composition can adversely affect biologicalactivity of that polypeptide, resulting in loss of therapeutic efficacyof the pharmaceutical composition. Furthermore, aggregate formation maycause other problems such as blockage of tubing, membranes, or pumpswhen the polypeptide-containing pharmaceutical composition isadministered using an infusion system.

A pharmaceutical composition may comprise an amount of an amino acidbase sufficient to decrease aggregate formation of the polypeptideduring storage of the composition. The term “amino acid base” refers toone or more amino acids (such as methionine, histidine, imidazole,arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), oranalogues thereof. Any amino acid may be present either in its free baseform or in its salt form. Any stereoisomer (i.e., L, D, or a mixturethereof) of the amino acid base may be present.

Methionine (or other sulphuric amino acids or amino acid analogous) maybe added to inhibit oxidation of methionine residues to methioninesulfoxide when the polypeptide acting as the therapeutic agent is apolypeptide comprising at least one methionine residue susceptible tosuch oxidation. Any stereoisomer of methionine (L or D) or combinationsthereof can be used.

A pharmaceutical composition may comprise a stabiliser selected from thegroup of high molecular weight polymers or low molecular compounds. Thestabiliser may e.g. be selected from polyethylene glycol (e.g. PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone,carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-Land HPMC), cyclodextrins, sulphur-containing substances asmonothioglycerol, thioglycolic acid and 2-methylthioethanol, anddifferent salts (e.g. sodium chloride). A pharmaceutical composition maycomprise additional stabilising agents such as, but not limited to,methionine and EDTA, which protect the polypeptide against methionineoxidation, and a nonionic surfactant, which protects the polypeptideagainst aggregation associated with freeze-thawing or mechanicalshearing.

A pharmaceutical composition may comprise one or more surfactants,preferably a surfactant, at least one surfactant, or two differentsurfactants. The term “surfactant” refers to any molecules or ions thatare comprised of a water-soluble (hydrophilic) part, and a fat-soluble(lipophilic) part. The surfactant may e.g. be selected from the groupconsisting of anionic surfactants, cationic surfactants, nonionicsurfactants, and/or zwitterionic surfactants.

A pharmaceutical composition may comprise one or more proteaseinhibitors, such as, e.g., EDTA (ethylenediamine tetraacetic acid),and/or benzamidineHCl.

Additional, optional, ingredients of a pharmaceutical compositioninclude, e.g., wetting agents, emulsifiers, antioxidants, bulkingagents, metal ions, oily vehicles, proteins (e.g., human serum albumin,gelatine), and/or a zwitterion (e.g., an amino acid such as betaine,taurine, arginine, glycine, lysine and histidine).

Still further, a pharmaceutical composition may be formulated as isknown in the art of oral formulations of insulinotropic compounds, e.g.using any one or more of the formulations described in WO 2008/145728.

An administered dose may contain from 0.01 mg-100 mg of the derivative,or from 0.01-50 mg, or from 0.01-20 mg, or from 0.01-10 mg of thederivative.

The derivative may be administered in the form of a pharmaceuticalcomposition. It may be administered to a patient in need thereof atseveral sites, for example, at topical sites such as skin or mucosalsites; at sites which bypass absorption such as in an artery, in a vein,or in the heart; and at sites which involve absorption, such as in theskin, under the skin, in a muscle, or in the abdomen.

The route of administration may be, for example, lingual; sublingual;buccal; in the mouth; oral; in the stomach; in the intestine; nasal;pulmonary, such as through the bronchioles, the alveoli, or acombination thereof; parenteral, epidermal; dermal; transdermal;conjunctival; uretal; vaginal; rectal; and/or ocular. A composition maybe an oral composition, and the route of administration is per oral.

A composition may be administered in several dosage forms, for exampleas a solution; a suspension; an emulsion; a microemulsion; multipleemulsions; a foam; a salve; a paste; a plaster; an ointment; a tablet; acoated tablet; a chewing gum; a rinse; a capsule such as hard or softgelatine capsules; a suppositorium; a rectal capsule; drops; a gel; aspray; a powder; an aerosol; an inhalant; eye drops; an ophthalmicointment; an ophthalmic rinse; a vaginal pessary; a vaginal ring; avaginal ointment; an injection solution; an in situ transformingsolution such as in situ gelling, setting, precipitating, and in situcrystallisation; an infusion solution; or as an implant. A compositionmay be a tablet, optionally coated, a capsule, or a chewing gum.

A composition may further be compounded in a drug carrier or drugdelivery system, e.g. in order to improve stability, bioavailability,and/or solubility. In a particular embodiment a composition may beattached to such system through covalent, hydrophobic, and/orelectrostatic interactions. The purpose of such compounding may be,e.g., to decrease adverse effects, achieve chronotherapy, and/orincrease patient compliance.

A composition may also be used in the formulation of controlled,sustained, protracting, retarded, and/or slow release drug deliverysystems.

Parenteral administration may be performed by subcutaneous,intramuscular, intraperitoneal, or intravenous injection by means of asyringe, optionally a pen-like syringe, or by means of an infusion pump.

A composition may be administered nasally in the form of a solution, asuspension, or a powder; or it may be administered pulmonally in theform of a liquid or powder spray.

Transdermal administration is a still further option, e.g. byneedle-free injection, from a patch such as an iontophoretic patch, orvia a transmucosal route, e.g. buccally.

A composition may be a stabilised formulation. The term “stabilisedformulation” refers to a formulation with increased physical and/orchemical stability, preferably both. In general, a formulation must bestable during use and storage (in compliance with recommended use andstorage conditions) until the expiration date is reached.

The term “physical stability” refers to the tendency of the polypeptideto form biologically inactive and/or insoluble aggregates as a result ofexposure to thermo-mechanical stress, and/or interaction withdestabilising interfaces and surfaces (such as hydrophobic surfaces).The physical stability of an aqueous polypeptide formulation may beevaluated by means of visual inspection, and/or by turbiditymeasurements after exposure to mechanical/physical stress (e.g.agitation) at different temperatures for various time periods.Alternatively, the physical stability may be evaluated using aspectroscopic agent or probe of the conformational status of thepolypeptide such as e.g. Thioflavin T or “hydrophobic patch” probes.

The term “chemical stability” refers to chemical (in particularcovalent) changes in the polypeptide structure leading to formation ofchemical degradation products potentially having a reduced biologicalpotency, and/or increased immunogenic effect as compared to the intactpolypeptide. The chemical stability can be evaluated by measuring theamount of chemical degradation products at various time-points afterexposure to different environmental conditions, e.g. by SEC-HPLC, and/orRP-HPLC.

The treatment with a derivative according to the present invention mayalso be combined with one or more additional pharmacologically activesubstances, e.g. selected from antidiabetic agents, antiobesity agents,appetite regulating agents, antihypertensive agents, agents for thetreatment and/or prevention of complications resulting from orassociated with diabetes and agents for the treatment and/or preventionof complications and disorders resulting from or associated withobesity. Examples of these pharmacologically active substances are:Insulin, sulphonylureas, biguanides, meglitinides, glucosidaseinhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV)inhibitors, inhibitors of hepatic enzymes involved in stimulation ofgluconeogenesis and/or glycogenolysis, glucose uptake modulators,compounds modifying the lipid metabolism such as antihyperlipidemicagents as HMG CoA inhibitors (statins), Gastric Inhibitory Polypeptides(GIP analogs), compounds lowering food intake, RXR agonists and agentsacting on the ATP-dependent potassium channel of the β-cells;Cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin,pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide,repaglinide; β-blockers such as alprenolol, atenolol, timolol, pindolol,propranolol and metoprolol, ACE (angiotensin converting enzyme)inhibitors such as benazepril, captopril, enalapril, fosinopril,lisinopril, alatriopril, quinapril and ramipril, calcium channelblockers such as nifedipine, felodipine, nicardipine, isradipine,nimodipine, diltiazem and verapamil, and α-blockers such as doxazosin,urapidil, prazosin and terazosin; CART (cocaine amphetamine regulatedtranscript) agonists, NPY (neuropeptide Y) antagonists, PYY agonists, Y2receptor agonists, Y4 receptor agonits, mixed Y2/Y4 receptor agonists,MC4 (melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosisfactor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP(corticotropin releasing factor binding protein) antagonists, urocortinagonists, β3 agonists, oxyntomodulin and analogues, MSH(melanocyte-stimulating hormone) agonists, MCH (melanocyte-concentratinghormone) antagonists, CCK (cholecystokinin) agonists, serotoninre-uptake inhibitors, serotonin and noradrenaline re-uptake inhibitors,mixed serotonin and noradrenergic compounds, 5HT (serotonin) agonists,bombesin agonists, galanin antagonists, growth hormone, growth hormonereleasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DAagonists (bromocriptin, doprexin), lipase/amylase inhibitors, RXR(retinoid X receptor) modulators, TR β agonists; histamine H3antagonists,

Gastric Inhibitory Polypeptide agonists or antagonists (GIP analogs),gastrin and gastrin analogs.

The treatment with a derivative according to this invention may also becombined with a surgery that influences the glucose levels, and/or lipidhomeostasis such as gastric banding or gastric bypass.

Pharmaceutical Indications

The present invention also relates to a derivative of the invention, foruse as a medicament.

In particular embodiments, the derivative of the inventionmay be usedfor the following medical treatments, all preferably relating one way orthe other to diabetes:

(i) prevention and/or treatment of all forms of diabetes, such ashyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetesof the young), gestational diabetes, and/or for reduction of HbA1 C;

(ii) delaying or preventing diabetic disease progression, such asprogression in type 2 diabetes, delaying the progression of impairedglucose tolerance (IGT) to insulin requiring type 2 diabetes, and/ordelaying the progression of non-insulin requiring type 2 diabetes toinsulin requiring type 2 diabetes;

(iii) improving β-cell function, such as decreasing β-cell apoptosis,increasing β-cell function and/or β-cell mass, and/or for restoringglucose sensitivity to β-cells;

(iv) prevention and/or treatment of cognitive disorders;

(v) prevention and/or treatment of eating disorders, such as obesity,e.g. by decreasing food intake, reducing body weight, suppressingappetite, inducing satiety; treating or preventing binge eatingdisorder, bulimia nervosa, and/or obesity induced by administration ofan antipsychotic or a steroid; reduction of gastric motility; and/ordelaying gastric emptying;

(vi) prevention and/or treatment of diabetic complications, such asneuropathy, including peripheral neuropathy; nephropathy; orretinopathy;

(vii) improving lipid parameters, such as prevention and/or treatment ofdyslipidemia, lowering total serum lipids; lowering HDL; lowering small,dense LDL; lowering VLDL: lowering triglycerides; lowering cholesterol;increasing HDL; lowering plasma levels of lipoprotein a (Lp(a)) in ahuman; inhibiting generation of apolipoprotein a (apo(a)) in vitroand/or in vivo;

(iix) prevention and/or treatment of cardiovascular diseases, such assyndrome X; atherosclerosis; myocardial infarction; coronary heartdisease; stroke, cerebral ischemia; an early cardiac or earlycardiovascular disease, such as left ventricular hypertrophy; coronaryartery disease; essential hypertension; acute hypertensive emergency;cardiomyopathy; heart insufficiency; exercise tolerance; chronic heartfailure; arrhythmia; cardiac dysrhythmia; syncopy; atheroschlerosis;mild chronic heart failure; angina pectoris; cardiac bypass reocclusion;intermittent claudication (atheroschlerosis oblitterens); diastolicdysfunction; and/or systolic dysfunction;

(ix) prevention and/or treatment of gastrointestinal diseases, such asinflammatory bowel syndrome; small bowel syndrome, or Crohn's disease;dyspepsia; and/or gastric ulcers;

(x) prevention and/or treatment of critical illness, such as treatmentof a critically ill patient, a critical illness poly-nephropathy (CIPNP)patient, and/or a potential CIPNP patient; prevention of criticalillness or development of CIPNP; prevention, treatment and/or cure ofsystemic inflammatory response syndrome (SIRS) in a patient; and/or forthe prevention or reduction of the likelihood of a patient sufferingfrom bacteraemia, septicaemia, and/or septic shock duringhospitalisation; and/or

(xi) prevention and/or treatment of polycystic ovary syndrome (PCOS).

In a particular embodiment, the indication is selected from the groupconsisting of (i)-(iii) and (v)-(iix), such as indications (i), (ii),and/or (iii); or indication (v), indication (vi), indication (vii),and/or indication (iix).

In another particular embodiment, the indication is (i). In a furtherparticular embodiment the indication is (v). In a still furtherparticular embodiment the indication is (iix).

The following indications are particularly preferred: Type 2 diabetes,and/or obesity.

PARTICULAR EMBODIMENTS

The following are particular embodiments of the invention:

1. A derivative of a GLP-1 analogue,

which analogue comprises a first K residue at a position correspondingto position 37 of GLP-1(7-37) (SEQ ID NO: 1), a second K residue at aposition corresponding to position 26 of GLP-1(7-37), and a maximum often amino acid modifications as compared to GLP-1(7-37), wherein thefirst K residue is designated K³⁷, and the second K residue isdesignated K²⁶,

which derivative comprises two albumin binding moieties attached to K²⁶and K³⁷, respectively, wherein

the albumin binding moiety comprises a protracting moiety selected fromChem. 1, Chem. 2, Chem. 3, and Chem. 4:HOOC—(CH₂)_(x)—CO—*  Chem. 1:HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:R¹—C₆H₄—(CH₂)_(z)—CO—*  Chem. 3:HOOC—C₄SH₂—(CH₂)_(w)—CO—*  Chem. 4:

in which x is an integer in the range of 6-18, y is an integer in therange of 3-17, z is an integer in the range of 1-5, R¹ is a group havinga molar mass not higher than 150 Da, and w is an integer in the range of6-18;

with the proviso that when the protracting moiety is Chem. 1, thealbumin binding moiety further comprises a linker of formula Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5;

or a pharmaceutically acceptable salt, amide, or ester thereof.

2. The derivative of embodiment 1,

wherein the GLP-1 analogue comprises a first K residue at a positioncorresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1), a second Kresidue at a position corresponding to position 26 of GLP-1(7-37), and amaximum of ten amino acid modifications as compared to GLP-1(7-37),wherein the first K residue is designated K³⁷, and the second K residueis designated K²⁶,

which derivative comprises two albumin binding moieties attached to K²⁶and K³⁷, respectively, wherein

the albumin binding moiety comprises a protracting moiety selected fromChem. 2, Chem. 3, and Chem. 4:HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:R¹—C₆H₄—(CH₂)_(z)—CO—*  Chem. 3:HOOC—C₄SH₂—(CH₂)_(w)—CO—*  Chem. 4:

in which y is an integer in the range of 3-17, z is an integer in therange of 1-5, R¹ is a group having a molar mass not higher than 150 Da,and w is an integer in the range of 6-18;

or a pharmaceutically acceptable salt, amide, or ester thereof.

3. The derivative of embodiment 2, wherein the albumin binding moietyfurther comprises a linker.

4. The derivative of embodiment 3, wherein the linker comprises i) a Gludi-radical; and/or ii) a linker of formula Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5.5. The derivative of embodiment 4, wherein the Glu di-radical isselected from Chem. 6, and/or Chem. 7:

preferably Chem. 6.6. The derivative of embodiment 1,

wherein the GLP-1 analogue comprises a first K residue at a positioncorresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1), a second Kresidue at a position corresponding to position 26 of GLP-1(7-37), and amaximum of ten amino acid modifications as compared to GLP-1(7-37),wherein the first K residue is designated K³⁷, and the second K residueis designated K²⁶,

which derivative comprises two albumin binding moieties attached to K²⁶and K³⁷, respectively, wherein

the albumin binding moiety comprises

i) a protracting moiety of formula Chem. 1:HOOC—(CH₂)_(x)—CO—*  Chem. 1:

in which x is an integer in the range of 6-18; and

ii) a linker of formula Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5; or a pharmaceutically acceptable salt, amide, or esterthereof.

7. The derivative of embodiment 1,

wherein the GLP-1 analogue comprises a first K residue at a positioncorresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1), a second Kresidue at a position corresponding to position 26 of GLP-1(7-37), and amaximum of ten amino acid modifications as compared to GLP-1(7-37),wherein the first K residue is designated K³⁷, and the second K residueis designated K²⁶;

which derivative comprises two protracting moieties attached to K²⁶ andK³⁷, respectively, via a linker, wherein

the protracting moiety is selected from Chem. 1, Chem. 2, Chem. 3, andChem. 4:HOOC—(CH₂)_(x)—CO—*  Chem. 1:HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:R¹—C₆H₄—(CH₂)_(z)—CO—*  Chem. 3:HOOC—C₄SH₂—(CH₂)_(w)—CO—*  Chem. 4:

in which x is an integer in the range of 6-18, y is an integer in therange of 3-17, z is an integer in the range of 1-5, R¹ is a group havinga molar mass not higher than 150 Da, and w is an integer in the range of6-18; and

the linker comprises Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5;

or a pharmaceutically acceptable salt, amide, or ester thereof.

8. The derivative of any one of embodiments 1-7, wherein Chem. 5 is afirst linker element.

9. The derivative of any one of embodiments 1-8, wherein k is 1.

10. The derivative of any one of embodiments 1-9, wherein n is 1.

11. The derivative of any one of embodiments 1-10, wherein Chem. 5 isincluded m times, wherein m is an integer in the range of 1-10.

12. The derivative of embodiment 11, wherein m is an integer in therange of 1-6; preferably in the range of 1-4; more preferably m is 1 or2; or most preferably m is 2.

13. The derivative of any one of embodiments 11-12, wherein, when m isdifferent from 1, the Chem. 5 elements are interconnected via amidebond(s).

14. The derivative of any one of embodiments 1-13, wherein the linkerconsists of one or more Chem. 5 elements.

15. The derivative of any one of embodiments 1-13, wherein the linkerfurther comprises a second linker element; preferably a Glu di-radical;more preferably selected from Chem. 6, and/or Chem. 7:

most preferably Chem. 6.16. The derivative of embodiment 15, wherein the Glu di-radical isincluded p times, wherein p is an integer in the range of 1-3.17. The derivative of embodiment 16, wherein p is 1, 2, or 3; preferably1 or 2; or most preferably 1.18. The derivative of any one of embodiments 1-17, wherein the Gludi-radical is a radical of L-Glu or D-Glu, preferably of L-Glu.19. The derivative of any one of embodiments 16-18, wherein the one ormore Glu di-radicals and the one or more Chem. 5 elements areinterconnected via amide bond(s).20. The derivative of any one of embodiments 1-19, wherein the linkercomprises a further linker element, such as a third linker element.21. The derivative of embodiment 20, wherein the third linker element is*—NH—(CH₂)_(q)—CHR²—CO—*,  Chem. 8:in which q is an integer in the range of 2-12, and R² is hydrogen (H),amino (NH₂), or a C₁—C₅ lower alcohol.22. The derivative of embodiment 21, wherein q is 4, 6, or 10.23. The derivative of any one of embodiments 21-22, wherein Chem. 8 is aradical of amino hexanoic acid, amino octanoic acid, amino dodecanoicacid, or lysine.24. The derivative of embodiment 23, wherein the radicalised amino groupis at the epsilon position.25. The derivative of any one of embodiments 1-24, wherein the linkerconsists of m times Chem. 5 and p times the Glu di-radical.26. The derivative of embodiment 25, wherein (m,p) is (2,2), (2,1),(2,3), (4,1), (6,1), (1,0), (1,1), (1,2), (0,1), or (0,2); preferably(2,1), (2,0), (1,0), (1,1), (0,1), or (0,2); more preferably (2,1),(2,2), or (1,2); even more preferably (1,1) or (2,1); or most preferably(2,1).27. The derivative of any one of embodiments 25-26, wherein the m Chem.5 elements and the p Glu di-radicals are interconnected via amide bonds.28. The derivative of any one of embodiments 21-24, wherein the linkerconsists of m times Chem. 5, p times the Glu di-radical, and Chem. 8.29. The derivative of embodiment 28, wherein (m,p) is (2,1), or (1,1);preferably (2,1).30. The derivative of any one of embodiments 28-29, wherein the m Chem.5 elements, the p Glu di-radicals, and the Chem. 8 element areinterconnected via amide bonds.31. The derivative of any one of embodiments 1-30, wherein the linkerand the protracting moiety are interconnected via an amide bond.32. The derivative of any one of embodiments 1-31, wherein the linkerand the GLP-1 analogue are interconnected via an amide bond.33. The derivative of embodiment 32, wherein the linker is attached tothe epsilon-amino group of K²⁶ or K³⁷.34. The derivative of any one of embodiments 1-33, wherein the linkerhas(i) from 5 to 41 C-atoms; preferably from 5-17 C-atoms; such as 5, 6,11, 12, or 17 C-atoms; for example 5, 6 or 12 C-atoms, or 11 or 17C-atoms; or most preferably 17 C-atoms;or(ii) from 5-30 C-atoms, preferably from 5-25 C-atoms, more preferablyfrom 5-20 C-atoms, or most prefarbly from 5-17 C-atoms.35. The derivative of any one of embodiments 1-34, wherein the linkerhas(i) from 4 to 28 hetero atoms; preferably from 4 to 12 hetero atoms;such as 4, 8, or 12 hetero atoms; more preferably 8 or 12 hetero atoms;or most preferably 12 hetero atoms; or(ii) from 4-20 hetero atoms, preferably from 4-18 hetero atoms, morepreferably from 4-14 hetero atoms, or most preferably from 4-12 heteroatoms.36. The derivative of embodiment 35, wherein the hetero atoms are N—,and/or O-atoms.37. The derivative of any one of embodiments 34-36, wherein the linkerhas from 1 to 7 N-atoms; preferably from 1 to 3 N-atoms; such as 1, 2,or 3 N-atoms; for example 1, or 2 N-atoms; or most preferably 3 N-atoms.38. The derivative of any one of embodiments 34-37, wherein the linkerhas from 3 to 21 O-atoms; preferably from 3 to 9 O-atoms; such as 3, 6,or 9 O-atoms; for example 3, or 6 O-atoms; or most preferably 9 O-atoms.39. The derivative of any one of embodiments 1-38, wherein the linkerconsists of two times Chem. 5, interconnected via an amide bond, andbeing connected at its *—NH end to the *—CO end of the protractingmoiety, and at its *—CO end to the epsilon amino group of K²⁶ or K³⁷ ofthe GLP-1 analogue.40. The derivative of any one of embodiments 1-38, wherein the linkerconsists of four times Chem. 5, interconnected via amide bonds, andconnected at its *—NH end to the *—CO end of the protracting moiety, andat its *—CO end to the epsilon amino group of K²⁶ or K³⁷ of the GLP-1analogue.41. The derivative of any one of embodiments 1-38, wherein the linkerconsists of two times Chem. 6 and two times Chem. 5, interconnected viaamide bonds and in the sequence indicated, the linker being connected atits *—NH end to the *—CO end of the protracting moiety, and at its *—COend to the epsilon amino group of K²⁶ or K³⁷ of the GLP-1 analogue.42. The derivative of any one of embodiments 1-38, wherein the linkerconsists of two times Chem. 5 and one time Chem. 6, interconnected viaamide bonds and in the sequence indicated, the linker being connected atits *—NH end to the *—CO end of the protracting moiety, and at its free*—CO end to the epsilon amino group of K²⁶ or K³⁷ of the GLP-1 analogue.43. The derivative of any one of embodiments 1-38, wherein the linkerconsists of three times Chem. 6 and two times Chem. 5, interconnectedvia amide bonds and in the sequence indicated, the linker beingconnected at its *—NH end to the *—CO end of the protracting moiety, andat its free *—CO end to the epsilon amino group of K²⁶ or K³⁷ of theGLP-1 analogue.44. The derivative of any one of embodiments 1-38, wherein the linkerconsists of one time Chem. 6 and two times Chem. 5, interconnected viaamide bonds and in the sequence indicated, the linker being connected atits *—NH end to the *—CO end of the protracting moiety, and at its *—COend to the epsilon amino group of K²⁶ or K³⁷ of the GLP-1 analogue.45. The derivative of any one of embodiments 1-38, wherein the linkerconsists of one time Chem. 6, one time Chem. 5, and one time Chem. 6,interconnected via amide bonds and in the sequence indicated, the linkerbeing connected at its *—NH end to the *—CO end of the protractingmoiety, and at its *—CO end to the epsilon amino group of K²⁶ or K³⁷ ofthe GLP-1 analogue.46. The derivative of any one of embodiments 1-38, wherein the linkerconsists of one time Chem. 6 and four times Chem. 5, interconnected viaamide bonds and in the sequence indicated, the linker being connected atits *—NH end to the *—CO end of the protracting moiety, and at its *—COend to the epsilon amino group of K²⁶ or K³⁷ of the GLP-1 analogue.47. The derivative of any one of embodiments 1-38, wherein the linkerconsists of one time Chem. 6 and six times Chem. 5, interconnected viaamide bonds and in the sequence indicated, the linker being connected atits *—NH end to the *—CO end of the protracting moiety and at its *—COend to the epsilon amino group of K²⁶ or K³⁷ of the GLP-1 analogue.48. The derivative of any one of embodiments 1-38, wherein the linkerconsists of one time Chem. 6 and one time Chem. 5, interconnected viaamide bonds and in the sequence indicated, the linker being connected atits *—NH end to the *—CO end of the protracting moiety, and at its *—COend to the epsilon amino group of K²⁶ or K³⁷ of the GLP-1 analogue.49. The derivative of any one of embodiments 1-38, wherein the linkerconsists of one time Chem. 5, one time Chem. 6, and one time Chem. 5,interconnected via amide bonds and in the sequence indicated, the linkerbeing connected at its *—NH end to the *—CO end of the protractingmoiety, and at its *—CO end to the epsilon amino group of K²⁶ or K³⁷ ofthe GLP-1 analogue.50. The derivative of any one of embodiments 1-38, wherein the linkerconsists of one time Chem. 7, and two times Chem. 5, interconnected viaamide bonds and in the sequence indicated, the linker being connected atits *—NH end to the *—CO end of the protracting moiety, and at its *—COend to the epsilon amino group of K²⁶ or K³⁷ of the GLP-1 analogue.51. The derivative of any one of embodiments 1-38, wherein the linkerconsists of one time Chem. 5, the linker being connected at its *—NH endto the *—CO end of the protracting moiety, and at its *—CO end to theepsilon amino group of K²⁶ or K³⁷ of the GLP-1 analogue.52. The derivative of any one of embodiments 1-38, wherein the linkerconsists of one time Chem. 6, the linker being connected at its *—NH endto the *—CO end of the protracting moiety, and at its *—CO end to theepsilon amino group of K²⁶ or K³⁷ of the GLP-1 analogue.53. The derivative of any one of embodiments 1-38, wherein the linkerconsists of two times Chem. 6, interconnected via amide bonds, thelinker being connected at its *—NH end to the *—CO end of theprotracting moiety, and at its *—CO end to the epsilon amino group ofK²⁶ or K³⁷ of the GLP-1 analogue.54. The derivative of any one of embodiments 1-53, wherein the linkerconsists of one time Chem. 6, one time Chem. 8, in which preferably q is10 and R² is H, one time Chem. 6, and two times Chem. 5, interconnectedvia amide bonds and in the sequence indicated, the linker beingconnected at its *—NH end to the *—CO end of the protracting moiety, andat its *—CO end to the epsilon amino group of K²⁶ or K³⁷ of the GLP-1analogue.55. The derivative of any one of embodiments 1-54, wherein the linkerconsists of one time Chem. 6, one time Chem. 8, in which preferably q is4 and R² is H, one time Chem. 6, and two times Chem. 5, interconnectedvia amide bonds and in the sequence indicated, the linker beingconnected at its *—NH end to the *—CO end of the protracting moiety, andat its *—CO end to the epsilon amino group of K²⁶ or K³⁷ of the GLP-1analogue.56. The derivative of any one of embodiments 1-55, wherein the linkerconsists of one time Chem. 6, one time Chem. 8, in which preferably q is6 and R² is H, one time Chem. 6, and two times Chem. 5, interconnectedvia amide bonds and in the sequence indicated, the linker beingconnected at its *—NH end to the *—CO end of the protracting moiety, andat its *—CO end to the epsilon amino group of K²⁶ or K³⁷ of the GLP-1analogue.57. The derivative of any one of embodiments 15-18, wherein the linkerconsists of one time Chem. 6, one time Chem. 8, in which preferably q is4 and R² is NH₂, one time Chem. 6, and two times Chem. 5, interconnectedvia amide bonds and in the sequence indicated, the linker beingconnected at its *—NH end to the *—CO end of the protracting moiety, andat its *—CO end to the epsilon amino group of K²⁶ or K³⁷ of the GLP-1analogue.58. The derivative of any one of embodiments 1-57, wherein theprotracting moiety is Chem. 1.59. The derivative of any one of embodiments 1-58, wherein x is an evennumber.60. The derivative of any one of embodiments 1-59, wherein x is aninteger in the range of 8-16, such as 8, 10, 12, 14, or 16; orpreferably in the range of 10-14.61. The derivative of any one of embodiments 1-60, wherein x is 10, 12,or 14; preferably 14; more preferably 10; or most preferably 12.62. The derivative of any one of embodiments 1-61, wherein Chem. 1 isrepresented by Chem. 1a:

where x is as defined in any one of embodiments 1-61.63. The derivative of any one of embodiments 1-57, wherein theprotracting moiety is Chem. 2.64. The derivative of any one of embodiments 1-63, wherein y is an oddnumber.65. The derivative of any one of embodiments 1-64, wherein y is aninteger in the range of 7-17, such as 7, 9, 11, 13, 15, or 17;preferably 7-15, such as, for example, 9, 11 or 15.66. The derivative of any one of embodiments 1-65, wherein y is 7, 8, 9,11, or 15.67. The derivative of any one of embodiments 1-66, wherein y is 7, 9,11, or 15.68. The derivative of any one of embodiments 1-67, wherein y is 7.69. The derivative of any one of embodiments 1-68, wherein y is 9.70. The derivative of any one of embodiments 1-69, wherein y is 11.71. The derivative of any one of embodiments 1-70, wherein y is 15.72. The derivative of any one of embodiments 1-71, wherein Chem. 2 isrepresented by Chem. 2a, or Chem. 2b:

preferably by Chem. 2a;wherein y is as defined in any one of embodiments 1-71.73. The derivative of any one of embodiments 1-57, wherein theprotracting moiety is Chem. 3.74. The derivative of any one of embodiments 1-73, wherein z is an oddnumber; preferably 3.75. The derivative of any one of embodiments 1-74, wherein R¹ is a grouphaving a molar mass not higher than 127 Da.76. The derivative of any one of embodiments 1-75, wherein R¹ is a grouphaving a molar mass in the range of 1-127 Da; preferably 1-125 Da, morepreferably 1-100 Da, even more preferably 1-75 Da, or most preferably1-50 Da.77. The derivative of any one of embodiments 1-76, wherein R¹ is a grouphaving(ii) a molar mass below 130 Da, preferably below 100 Da, more preferablybelow 75 Da, even more preferably below 60 Da, or most preferably below50 Da; or(iii) a molar mass below 40 Da, preferably below 30 Da, more preferablybelow 20 Da, or most preferably below 15 Da.78. The derivative of any one of embodiments 1-77, wherein R¹ is —H.79. The derivative of any one of embodiments 1-78, wherein R¹ is ahalogen radical.80. The derivative of any one of embodiments 1-79, wherein R¹ is —I.81. The derivative of any one of embodiments 1-80, wherein R¹ is linearor branched C1-C5 alkyl; preferably C1-C4 alkyl; more preferably methyl;or most preferably tert. butyl.82. The derivative of any one of embodiments 1-81, wherein Chem. 3 isrepresented by Chem. 3a:

wherein R¹ and z are as defined in any one of embodiments 1-81.83. The derivative of any one of embodiments 1-57, wherein theprotracting moiety is Chem. 4.84. The derivative of any one of embodiments 1-83, wherein w is an evennumber.85. The derivative of any one of embodiments 1-84, wherein w is aninteger in the range of 8-16; or preferably in the range of 10-14.86. The derivative of any one of embodiments 1-85, wherein w is 10, 12,or 14; preferably 14; more preferably 10; or most preferably 12.87. The derivative of any one of embodiments 1-86, wherein Chem. 4 isrepresented by Chem. 4a:

wherein w is as defined in any one of embodiments 1-86.88. The derivative of any one of embodiments 1-87, wherein the twoprotracting moieites are substantially identical; such as at least 80%,at least 85%, at least 90%, at least 95%, or at least 99% identical.89. The derivative of any one of embodiments 1-88, wherein the twoprotracting moieties have a similarity of at least 0.5; preferably atleast 0.6; more preferably at least 0.7, or at least 0.8; even morepreferably at least 0.9; or most preferably at least 0.99, such as asimilarity of 1.0.90. The derivative of any one of embodiments 1-89, wherein the twolinkers are substantially identical; such as at least 80%, at least 85%,at least 90%, at least 95%, or at least 99% identical.91. The derivative of any one of embodiments 1-90, wherein the twolinkers have a similarity of at least 0.5; preferably at least 0.6; morepreferably at least 0.7, or at least 0.8; even more preferably at least0.9; or most preferably at least 0.99, such as a similarity of 1.0.92. The derivative of any one of embodiments 1-91, wherein the twoalbumin binders, such as the two side chains consisting of protractingmoiety and linker, are substantially identical; such as at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% identical.93. The derivative of any one of embodiments 1-92, wherein the twoalbumin binders, such as the two side chains consisting of protractingmoiety and linker, have a similarity of at least 0.5; preferably atleast 0.6; more preferably at least 0.7, or at least 0.8; even morepreferably at least 0.9; or most preferably at least 0.99, such as asimilarity of 1.0.94. The derivative of any one of embodiments 88-93, wherein the twochemical structures to be compared are represented as fingerprints, suchas a) ECFP_(—)6 fingerprints; b) UNITY fingerprints; and/or c) MDLfingerprints; and wherein for each of a), b) and c) the Tanimotocoefficient is preferably used for calculating the similarity, oridentity, of the two fingerprints.95. The derivative of any one of embodiments 1-94, wherein

a) the positions corresponding to position 37 and 26 of GLP-1(7-37) (SEQID NO: 1), and/or

b) the number of amino acid modifications as compared to GLP-1(7-37)(SEQ ID NO: 1)

are identified by handwriting and eyeballing.

96. The derivative of any one of embodiments 1-95, wherein

a) the positions corresponding to position 37 and 26 of GLP-1(7-37) (SEQID NO: 1), and/or

b) the number of amino acid modifications as compared to GLP-1(7-37)(SEQ ID NO: 1)

are identified by use of a standard protein or peptide alignmentprogram.

97. The derivative of embodiment 96, wherein the alignment program is aNeedleman-Wunsch alignment.

98. The derivative of any one of embodiments 96-97, wherein the defaultscoring matrix and the default identity matrix is used.

99. The derivative of any one of embodiments 96-98, wherein the scoringmatrix is BLOSUM62.

100. The derivative of any one of embodiments 96-99, wherein the penaltyfor the first residue in a gap is −10 (minus ten).

101. The derivative of any one of embodiments 96-100, wherein thepenalties for additional residues in a gap is −0.5 (minus point five).

102. The derivative of any one of embodiments 1-101, wherein theanalogue comprises no K residues other than the first and the second Kresidue.

103. The derivative of any one of embodiments 1-102, wherein the aminoacid modification(s) is (are) at one or more positions corresponding tothe following positions in GLP-1(7-37) (SEQ ID NO: 1): 7, 8, 9, 23, 30,31, 34, 36, 37, and 38.

104. The derivative of any one of embodiments 1-103, wherein theanalogue comprises, preferably has, a minimum of two amino acidmodifications, as compared to GLP-1(7-37) (SEQ ID NO: 1); the minimumtwo amino acid modifications being preferably at each of the positionscorresponding to position 34 and 37 of GLP-1(7-37) (SEQ ID NO: 1), andmore preferably so that the amino acid at the position corresponding toposition 37 is K, and the amino acid at the position corresponding toposition 34 is not K.105. The derivative of any one of embodiments 1-104, wherein the GLP-1analogue has a C-terminal amide.106. The derivative of embodiment 105, wherein the amino acid at theposition corresponding to position 34 is R or Q.107. The derivative of any one of embodiments 1-106, wherein the aminoacid modifications are selected from the following: (R³⁴ or Q³⁴), K³⁷,(Des⁷ or Imp⁷), (D-Ala⁸, Des⁸, Aib⁸, G⁸, or S⁸), (Q⁹ or G⁹), R²³, E³⁰,H³¹, G³⁶, and/or (E³⁸ or G³⁸).108. The derivative of any one of embodiments 1-107, wherein the aminoacid modifications are selected from the following: (R³⁴ or Q³⁴), K³⁷,(Des⁷ or Imp⁷), (Des⁸ or Aib⁸), (Q⁹ or G⁹), R²³, E³⁰, H³¹, G³⁶, and/or(E³⁸ or G³⁸).109. The derivative of any one of embodiments 1-108, wherein theanalogue comprises (R³⁴ or Q³⁴), and K³⁷.110. The derivative of any one of embodiments 1-109, wherein theanalogue comprises Imp⁷, and/or (Aib⁸ or S⁸); preferably Imp⁷, and/orAib⁸; more preferably Imp⁷; or most preferably Aib⁸.111. The derivative of any one of embodiments 1-110, wherein theanalogue comprises G³⁸ or E³⁸, preferably E³⁸.112. The derivative of any one of embodiments 1-111, wherein theanalogue comprises Q⁹ or G⁹.113. The derivative of any one of embodiments 1-112, wherein theanalogue comprises G³⁶.114. The derivative of any one of embodiments 1-113, wherein theanalogue comprises H³¹.115. The derivative of any one of embodiments 1-114, wherein theanalogue comprises R²³.116. The derivative of any one of embodiments 1-115, wherein theanalogue comprises des⁷ and/or des⁸, preferably both.117. The derivative of any one of embodiments 1-116, wherein one aminoacid has been deleted at a position corresponding to position 7 ofGLP-1(7-37) (SEQ ID NO: 1).118. The derivative of any one of embodiments 1-117, wherein one aminoaicd has been deleted at a position corresponding to position 8 ofGLP-1(7-37) (SEQ ID NO: 1).119. The derivative of any one of embodiments 1-118, wherein two aminoacids have been deleted at positions corresponding to position 7 and 8of GLP-1(7-37) (SEQ ID NO: 1).120. The derivative of any one of embodiments 1-119, which is ananalogue of GLP-1(8-37) (amino acids 2-31 of SEQ ID NO: 1), having up toten, nine, eight, or six amino acid modifications as compared toGLP-1(7-37) (SEQ ID NO: 1).121. The derivative of any one of embodiments 1-120, which is ananalogue of GLP-1(9-37) (amino acids 3-31 respectively, of SEQ ID NO:1), having up to ten, nine, eight, or six amino acid modifications ascompared to GLP-1(7-37) (SEQ ID NO: 1). 122. The derivative of any oneof embodiments 1-121, wherein the GLP-1 analogue corresponds to (a)K³⁷-GLP-1(7-37), (b) K³⁷-GLP-1(8-37), (c) K³⁷-GLP-1(9-37), or (d) ananalogue of any one of (a)-(c) having up to ten, nine, eight, or sixamino acid modifications as compared to GLP-1(7-37) (SEQ ID NO: 1).123. The derivative of any one of embodiments 1-122, wherein aHis-mimetic other than His is at a position corresponding to position 2of GLP-1(7-37) (SEQ ID NO: 1).124. The derivative of any one of embodiments 1-123, wherein aHis-Ala-mimetic other than His-Ala is at the positions corresponding toposition 7 and 8 of GLP-1(7-37) (SEQ ID NO: 1).125. The derivative of any one of embodiments 123-124, wherein theHis-mimetic, or the His-Ala mimetic, comprises a) imidazole; or b)pyridine.126. The derivative of embodiment 125, wherein the imidazole is aderivative of an imidazole which comprises a *—CO end, for covalentcoupling to *—NH of the N-terminal amino acid of the analogue, viaformation of an amide bond.127. The derivative of embodiment 125, wherein the pyridine is aderivative of pyridine which comprises a *—CO end, for covalent couplingto *—NH of the N-terminal amino acid of the analogue, via formation ofan amide bond.128. The derivative of any one of embodiments 125-127, wherein theimidazole derivative is mono-substituted.129. The derivative of any one of embodiments 125-127, wherein thepyridine derivative is mono-substituted.130. The derivative of any one of embodiments 125-129, wherein theimidazole derivative is substituted with a group comprising a carboxylicacid radical of a lower alkyl having from one to six carbon atoms.131. The derivative of any one of embodiments 125-129, wherein thepyridine derivative is substituted with a group comprising a carboxylicacid radical of a lower alkyl having from one to six carbon atoms.132. The derivative of any one of embodiments 130-131, wherein thecarboxylic acid radical is selected from acetyl; and straight orbranched propionyl, butyryl, pentanoyl; preferably acetyl.133. The derivative of any one of embodiments 1-132, wherein the aminoacid residue at the position corresponding to position 8 of GLP-1(7-37)(SEQ ID NO: 1) has 3H-Imidazol-4-yl-acetyl attached to its N-atom.134. The derivative of any one of embodiments 1-133, wherein the aminoacid residue at the position corresponding to position 8 of SEQ ID NO: 1is alanine.135. The derivative of any one of embodiments 125-134, wherein theimidazole is substituted with (methylcarbamoyl)-2-methyl-propionyl,(ethylcarbamoyl)-2-methyl-propionyl,(propylcarbamoyl)-2-methyl-propionyl, or(butylcarbamoyl)-2-methyl-propionyl; preferably with(ethylcarbamoyl)-2-methyl-propionyl.136. The derivative of any one of embodiments 1-135, wherein the aminoacid residue at the position corresponding to position 9 of GLP-1(7-37)(SEQ ID NO: 1) has{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl} attached toits N-atom.137. The derivative of any one of embodiments 125-136, wherein thepyridine is substituted with (methylcarbamoyl)-2-methyl-propionyl,(ethylcarbamoyl)-2-methyl-propionyl,(propylcarbamoyl)-2-methyl-propionyl, or(butylcarbamoyl)-2-methyl-propionyl; preferably with(methylcarbamoyl)-2-methyl-propionyl.138. The derivative of any one of embodiments 1-137, wherein the aminoacid residue at the position corresponding to position 9 of GLP-1(7-37)(SEQ ID NO: 1) has[2,2-dimethyl-3-oxo-3-(pyridin-2-ylmethylamino)propanoyl] attached toits N-atom.139. The derivative of any one of embodiments 1-138, wherein the aminoacid residue at the position corresponding to position 9 of the GLP-1analogue is glutamic acid.140. The derivative of any one of embodiments 1-139, wherein theanalogue does not comprise (H³¹ and Q³⁴).141. The derivative of any one of embodiments 1-140, wherein theanalogue does not comprise (des⁷ and des⁸); and/or does not comprise aHis-mimetic, or a His-Ala mimetic as defined in any one of embodiments116-140.142. The derivative of any one of embodiments 1-141, wherein theanalogue is an analogue of GLP-1(7-37), or GLP-1(9-37).143. The derivative of any one of embodiments 1-142, wherein theanalogue comprises, preferably has, the following amino acid changes, ormodifications, as compared to GLP-1(7-37) (SEQ ID NO: 1): i) (34R, 37K);ii) (8Aib, 34R, 37K); iii) (31H, 34Q, 37K); iv) (des7, des8, 34R, 37K),and optionally 38E; or v) (34R, 36G, 37K).144. The derivative of any one of embodiments 1-143, wherein theanalogue has a maximum of nine amino acid modifications.145. The derivative of any one of embodiments 1-144, wherein theanalogue has a maximum of eight amino acid modifications.146. The derivative of any one of embodiments 1-145, wherein theanalogue has a maximum of seven amino acid modifications.147. The derivative of any one of embodiments 1-146, wherein theanalogue has a maximum of six amino acid modifications.148. The derivative of any one of embodiments 1-147, wherein theanalogue has a maximum of five amino acid modifications.149. The derivative of any one of embodiments 1-148, wherein theanalogue has a maximum of four amino acid modifications.150. The derivative of any one of embodiments 1-149, wherein theanalogue has a maximum of three amino acid modifications.151. The derivative of any one of embodiments 1-150, wherein theanalogue has a maximum of two amino acid modifications.152. The derivative of any one of embodiments 1-151, wherein theanalogue has a minimum of two amino acid modifications.153. The derivative of any one of embodiments 1-152, wherein theanalogue has a minimum of three amino acid modifications.154. The derivative of any one of embodiments 1-153, wherein theanalogue has a minimum of four amino acid modifications.155. The derivative of any one of embodiments 1-154, wherein theanalogue has a minimum of five amino acid modifications.156. The derivative of any one of embodiments 1-155, wherein theanalogue has a minimum of six amino acid modifications.157. The derivative of any one of embodiments 1-156, wherein theanalogue has a minimum of seven amino acid modifications.158. The derivative of any one of embodiments 1-157, wherein theanalogue has a minimum of eight amino acid modifications.159. The derivative of any one of embodiments 1-158, wherein theanalogue has a minimum of nine amino acid modifications.160. The derivative of any one of embodiments 1-159, wherein theanalogue has a minimum of ten amino acid modifications.161. The derivative of any one of embodiments 1-160, wherein theanalogue has one amino acid modification.162. The derivative of any one of embodiments 1-161, wherein theanalogue has two amino acid modifications.163. The derivative of any one of embodiments 1-162, wherein theanalogue has three amino acid modifications.164. The derivative of any one of embodiments 1-163, wherein theanalogue has four amino acid modifications.165. The derivative of any one of embodiments 1-164, wherein theanalogue has five amino acid modifications.166. The derivative of any one of embodiments 1-165, wherein theanalogue has six amino acid modifications.167. The derivative of any one of embodiments 1-166, wherein theanalogue has seven amino acid modifications.168. The derivative of any one of embodiments 1-167, wherein theanalogue has eight amino acid modifications.169. The derivative of any one of embodiments 1-169, wherein theanalogue has nine amino acid modifications.170. The derivative of any one of embodiments 1-170, wherein theanalogue has ten amino acid modifications.171. The derivative of any one of embodiments 1-171, wherein themodifications are, independently, substitutions, additions, and/ordeletions.172. The derivative of any one of embodiments 1-172, wherein themodifications are substitutions.173. The derivative of any one of embodiments 1-173, wherein themodifications are deletions.174. The derivative of any one of embodiments 1-174, wherein themodifications are additions.175. The derivative of any one of embodiments 1-174, wherein

a) the position corresponding to any of the indicated positions ofGLP-1(7-37) (SEQ ID NO: 1), and/or

b) the number of amino acid modifications as compared to GLP-1(7-37)(SEQ ID NO: 1)

is/are identified by handwriting and eyeballing.

176. The derivative of any one of embodiments 1-175, wherein

a) the position corresponding to any of the indicated positions ofGLP-1(7-37) (SEQ ID NO: 1), and/or b) the number of amino acidmodifications as compared to GLP-1(7-37) (SEQ ID NO: 1)

is/are identified as described in any one of embodiments 96-101.

177. A compound selected from the following: Chem. 20, Chem. 21, Chem.22, Chem. 23, Chem. 24, Chem. 25, Chem. 26, Chem. 27, Chem. 28, Chem.29, Chem. 30, Chem. 31, Chem. 32, Chem. 33, Chem. 34, Chem. 35, Chem.36, Chem. 37, Chem. 38, Chem. 39, Chem. 40, Chem. 41, Chem. 42, Chem.43, Chem. 44, Chem. 45, Chem. 46, Chem. 47, Chem. 48, Chem. 49, Chem.50, Chem. 51, Chem. 52, Chem. 53, Chem. 54, Chem. 55, Chem. 56, Chem.57, Chem. 58, Chem. 59, Chem. 60, Chem. 61, Chem. 62, Chem. 63, Chem.64, Chem. 65, Chem. 66, Chem. 67, and Chem. 68; or a pharmaceuticallyacceptable salt, amide, or ester thereof.178. A compound characterised by its name, and selected from a listingof each of the names of the compounds of Examples 1-49 herein, or apharmaceutically acceptable salt, amide, or ester thereof.179. The compound of embodiment 178, which is a compound of embodiment177.180. The compound of any one of embodiments 178 and 179, which is aderivative according to any one of embodiments 1-176.181. The derivative of any one of embodiments 1-180 which is selectedfrom the following:

-   (i)    N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],    N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg³⁴,Gly³⁶,Lys³⁷]-GLP-1-(7-37)-peptide    (SEQ ID NO: 6),

-   N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[8-(4-carboxyphenoxy)octanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],    N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[8-(4-carboxyphenoxy)octanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide    (SEQ ID NO: 7),

-   N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],    N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,His³¹,Gln³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 3),

-   N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],    N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Gln⁹,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide    (SEQ ID NO: 8),

-   N^(ε26{)2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy)acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}-[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7),

-   N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[9-(4-carboxyphenoxy)nonanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],    N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[9-(4-carboxyphenoxy)nonanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide    (SEQ ID NO: 9),

-   N^(ε26)-[2-(2-[2-(2-[2-(2-[4-(10-(4-Carboxyphenoxy)decanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]-N^(ε37)-[2-(2-[2-(2-[2-(2-[4-(10-(4-Carboxyphenoxy)decanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)    ethoxy]ethoxy)acetyl][Aib⁸,His³¹,Gln³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 3),

-   N^(ε26)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[10-(4-carboxyphenoxy)decanoylamino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl],    N^(ε37)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[10-(4-carboxyphenoxy)decanoylamino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl][His³¹,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide    (SEQ ID NO: 10),

-   N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],    N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arq34,Lys37]-GLP-1-(7-37)-peptide    (SEQ ID NO: 9),

-   N^(ε26)-[(S)-4-Carboxy-4-{2-[2-(2-[2-(2-{2-[(13-carboxytridecanoylamino)]ethoxy}ethoxy)acetylamino]ethoxy)ethoxy]acetylamino}butyryl],    N^(ε37)-[(S)-4-Carboxy-4-{2-[2-(2-[2-(2-{2-[(13-carboxytridecanoylamino)]ethoxy}ethoxy)acetylamino]ethoxy)ethoxy]acetylamino}butyryl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7),

-   N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],    N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl][Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptidyl-Glu    (SEQ ID NO: 9), and

-   (ii)    N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methylpropionyl}-N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-[4-(4-tert-Butylphenyl)butyrylamino]-4-carboxybutyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-[4-(4-tert-Butylphenyl)butyrylamino]-4-carboxybutyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}[Arg³⁴,    Lys³⁷]GLP-1(9-37)-peptide (SEQ ID NO: 9),

-   N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],    N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Arg²³,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide    (SEQ ID NO: 4),

-   N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],    N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7),

-   N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[4-(4-iodo-phenyl)-butyrylamino]-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[4-(4-iodo-phenyl)-butyrylamino]-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7), and

-   (iii)    N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[12-(3-carboxyphenoxy)dodecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]a    cetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[12-(3-carboxyphenoxy)dodecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]a    cetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide (SEQ ID NO: 7),

-   N²⁶-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytndecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],    N^(ε37)-[2-(2-{2-[2-(2-{2-RS)-4-Carboxy-4-(13-carboxytndecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7),

-   N^(ε26)-[(S)-4-Carboxy-4-{2-[2-(2-[2-(2-{2-[(13-carboxytridecanoylamino)]ethoxy}ethoxy)acetylamino]ethoxy)ethoxy]acetylamino}butyryl],    N^(ε37)-[(S)-4-Carboxy-4-{2-[2-(2-[2-(2-{2-[(13-carboxytridecanoylamino)]ethoxy}ethoxy)acetylamino]ethoxy)ethoxy]acetylamino}butyryl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7),

-   N^(ε26{)2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}-[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7),

-   N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],    N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide    (SEQ ID NO: 9), and

-   (iv)    N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[12-(3-carboxyphenoxy)dodecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]a    cetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[12-(3-carboxyphenoxy)dodecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]a    cetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide (SEQ ID NO: 7),

-   N^(ε26{)2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}-[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7),

-   N^(ε26)-[2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]],    N^(ε37)-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    amide (SEQ ID NO: 7),

-   N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[4-(4-iodo-phenyl)-butyrylamino]-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[4-(4-iodo-phenyl)-butyrylamino]-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7), and

or a pharmaceutically acceptable salt, amide, or ester of any of thesecompounds.182. The derivative of embodiment 181, which is Chem. 62, or apharmaceutically acceptable salt, amide, or ester thereof.183. The derivative of embodiment 181, which is Chem. 40, or apharmaceutically acceptable salt, amide, or ester thereof.184. The derivative of embodiment 181, which is Chem. 21, or apharmaceutically acceptable salt, amide, or ester thereof.185. The derivative of embodiment 181, which is Chem. 55, or apharmaceutically acceptable salt, amide, or ester thereof.186. The derivative of embodiment 181, which is Chem. 51, or apharmaceutically acceptable salt, amide, or ester thereof.187. The derivative of embodiment 181, which is Chem. 44, or apharmaceutically acceptable salt, amide, or ester thereof.188. The derivative of embodiment 181, which is Chem. 46, or apharmaceutically acceptable salt, amide, or ester thereof.189. The derivative of embodiment 181, which is Chem. 31, or apharmaceutically acceptable salt, amide, or ester thereof.190. The derivative of embodiment 181, which is Chem. 35, or apharmaceutically acceptable salt, amide, or ester thereof.191. The derivative of embodiment 181, which is Chem. 23, or apharmaceutically acceptable salt, amide, or ester thereof.192. The derivative of any one of embodiments 1-191, which has GLP-1activity.193. The derivative of embodiment 192, wherein GLP-1 activity refers tothe capability of activating the human GLP-1 receptor.194. The derivative of embodiment 193, wherein activation of the humanGLP-1 receptor is measured in an in vitro assay, as the potency of cAMPproduction.195. The derivative of any one of embodiments 1-194, which has a potencycorresponding to an EC₅₀,a) below 10000 μM, more preferably below 5000 μM, even more preferablybelow 4000 μM, or most preferably below 3000 μM;b) at or below 3000 μM, preferably below 3000 μM, more preferably below2500 μM, even more preferably below 2000 μM, or most preferably below1500 μM;c) below 2000 μM, preferably below 1000 μM, more preferably below 800μM, even more preferably below 600 μM, or most preferably below 500 μM;c) below 400 μM, preferably below 300 μM, more preferably below 200 μM,even more preferably below 150 μM, or most preferably below 100 μM;d) below 80 μM, preferably below 60 μM, more preferably below 50 μM,even more preferably below 40 μM, or most preferably below 30 μM; orwhich has a potency corresponding to an EC₅₀,e) which is less than 10 times the EC₅₀ of semaglutide, preferably lessthan 8 times the EC₅₀ of semaglutide, more preferably less than 6 timesthe EC₅₀ of semaglutide, even more preferably less than 4 times the EC₅₀of semaglutide, or most preferably less than 2 times the EC₅₀, ofsemaglutide;f) which is less than 10 times the EC₅₀ of liraglutide, preferably lessthan 8 times the EC₅₀ of liraglutide, more preferably less than 6 timesthe EC₅₀ of liraglutide, even more preferably less than 4 times theEC₅₀, of liraglutide, or most preferably less than 2 times the EC₅₀, ofliraglutide; org) which is less than the EC₅₀ of liraglutide, preferably less than 0.8times the EC₅₀ of liraglutide, more preferably less than 0.6 times theEC₅₀ of liraglutide, even more preferably less than 0.5 times the EC₅₀of liraglutide, or most preferably less than or at 0.4 times the EC₅₀ ofliraglutide.196. The derivative of any one of embodiments 1-195, wherein the potencyis determined as EC₅₀, for the dose-response curve showingdose-dependent formation of cAMP in a medium containing the human GLP-1receptor, preferably using a stable transfected cell-line such asBHK467-12A (tk-ts13), and/or using for the determination of cAMP afunctional receptor assay, e.g. based on competition betweenendogenously formed cAMP and exogenously added biotin-labelled cAMP, inwhich assay cAMP is more preferably captured using a specific antibody,and/or wherein an even more preferred assay is the AlphaScreen cAMPAssay, most preferably the one described in Example 50.197. The derivative of any one of embodiments 1-196, for which the ratio[GLP-1 receptor binding affinity (IC₅₀) in the presence of 2.0% HSA(high albumin), divided by GLP-1 receptor binding affinity (IC₅₀) in thepresence of 0.005% HSA (low albumin)] is:a) at least 0.5, preferably at least 1.0, more preferably at least 10,even more preferably at least 20, or most preferably at least 30;b) at least 40, preferably at least 50, more preferably at least 60,even more preferably at least 70, or most preferably at least 80;c) at least 90, preferably at least 100, more preferably at least 200,still more preferably at least 300, even more preferably at least 400,or most preferably at least 500;d) at least 120, preferably at least 140, even more preferably at least160, or most preferably at least 180;e) at least 20% of the ratio of semaglutide, preferably at least 50% ofthe ratio of semaglutide, more preferably at least 75% of the ratio ofsemaglutide, or most preferably at least equal to the ratio ofsemaglutide; orf) at least equal to the ratio of liraglutide, preferably at least twicethe ratio of liraglutide, more preferably at least three times the ratioof liraglutide, even more preferably at least 4 times the ratio ofliraglutide, or most preferably at least 5 times the ratio ofliraglutide.198. The derivative of any one of embodiments 1-197, for which the GLP-1receptor binding affinity (IC₅₀) in the presence of 0.005% HSA (lowalbumin) isa) below 1000.00 nM, preferably below 600.00 nM, more preferably below100.00 nM, or most preferably below 50.00 nM; orb) below 20.00 nM, preferably below 10.00 nM, more preferably below 5.00nM, even more preferably below 2.00 nM, or most preferably below 1.00nM.199. The derivative of any one of embodiments 1-198, for which the GLP-1receptor binding affinity (IC₅₀) in the presence of 2.0% HSA (highalbumin) isa) below 1100.00 nM, preferably at or below 1000.00 nM, more preferablybelow 800.00 nM, or most prefererably below 600 nM; orb) below 400.00 nM, preferably below 300.00 nM, more preferably below200.00 nM, even more preferably below 100.00 nM, or most preferablybelow 50.00 nM.200. The derivative of any one of embodiments 1-199, wherein the bindingaffinity to the GLP-1 receptor is measured by way of displacement of¹²⁵I-GLP-1 from the receptor, preferably using a SPA binding assay.201. The derivative of any one of embodiments 1-200, wherein the GLP-1receptor is prepared using a stable, transfected cell line, preferably ahamster cell line, more preferably a baby hamster kidney cell line, suchas BHK tk-ts13.202. The derivative of any one of embodiments 1-201, wherein the IC₅₀value is determined as the concentration which displaces 50% of¹²⁵I-GLP-1 from the receptor.203. The derivative of any one of embodiments 1-202, which has an oralbioavailability, preferably an absolute oral bioavailability, which ishigher than that of semaglutide.204. The derivative of embodiment 203, wherein oral bioavailability ismeasured in vivo in rats, as exposure in plasma after direct injectioninto the intestinal lumen.205. The derivative of any one of embodiments 1-204, for which theplasma concentration (pM) of the derivative, determined 30 minutes afterinjection of a solution of the derivative in the jejunum of rat, dividedby the concentration (μM) of the injected solution (dose-correctedexposure at 30 min) is at least 40, preferably at least 50, morepreferably at least 60, still more preferably at least 70, even morepreferably at least 80, or most preferably at least 100.206. The derivative of any one of embodiments 1-205, for which theplasma concentration (pM) of the derivative, determined 30 minutes afterinjection of a solution of the derivative in the jejunum of rat, dividedby the concentration (μM) of the injected solution (dose-correctedexposure at 30 min) is at least 110, preferably at least 120, morepreferably at least 130, still more preferably at least 140, even morepreferably at least 150, or most preferably at least 160.207. The derivative of any one of embodiments 1-206, for which theplasma concentration (pM) of the derivative, determined 30 minutes afterinjection of a solution of the derivative in the jejunum of rat, dividedby the concentration (μM) of the injected solution (dose-correctedexposure at 30 min) is at least 180, preferably at least 190, morepreferably at least 200, or most preferably at least 210.208. The derivative of any one of embodiments 1-207, wherein the GLP-1derivative is tested in a concentration of 1000 uM in admixture with 55mg/ml sodium caprate.209. The derivative of any one of embodiments 1-208, wherein maleSprague Dawley rats are used, preferably with a body weight upon arrivalof approximately 240 g.210. The derivative of any one of embodiments 1-209, wherein the ratsare fasted for approximately 18 hours before the experiment.211. The derivative of any one of embodiments 1-210, wherein the ratsare taken into general anaesthesia after having fasted and before theinjection of the derivative in the jejunum.212. The derivative of any one of embodiments 1-211, wherein thederivative is administered in the proximal part of the jejunum (10 cmdistal for the duodenum) or in the mid-intestine (50 cm proximal for thececum).213. The derivative of any one of embodiments 1-212, wherein 100 μl ofthe derivative is injected into the jejunal lumen through a catheterwith a 1 ml syringe, and subsequently 200 μl of air is pushed into thejejunal lumen with another syringe, which is then left connected to thecatheter to prevent flow back into the catheter.214. The derivative of any one of embodiments 1-213, wherein bloodsamples (200 ul) are collected into EDTA tubes from the tail vein atdesired intervals, such as at times 0, 10, 30, 60, 120 and 240 min, andcentrifuged 5 minutes, 10000G, at 4° C. within 20 minutes.215. The derivative of any one of embodiments 1-214, wherein plasma(75u1) is separated, immediately frozen, and kept at −20° C. untilanalyzed for plasma concentration of the derivative.216. The derivative of any one of embodiments 1-215, wherein LOCI(Luminescent Oxygen Channeling Immunoassay) is used for analyzing theplasma concentration of the derivative.217. The derivative of any one of embodiments 1-216, wherein thederivative is effective at lowering blood glucose in vivo in db/db mice.218. The derivative of any one of embodiments 1-217, wherein thederivative is effective at lowering body weight in vivo in db/db mice.219. The derivative of any one of embodiments 1-218, wherein db/db miceare treated, s.c., with a suitable range of doses of the GLP-1derivative, and blood glucose and/or bodyweight is/are determined atappropriate intervals.220. The derivative of any one of embodiments 1-219, wherein the dose ofthe GLP-1 derivative is 0.3 nmol/kg, 1.0 nmol/kg, 3.0 nmol/kg, 10nmol/kg, 30 nmol/kg, and 100 nmol/kg, wherein kg refers to the bodyweight of the mouse.221. The derivative of any one of embodiments 1-220, wherein a controlgroup is treated with vehicle, s.c., preferably the medium in which theGLP-1 derivative is dissolved, e.g. with the following composition: 50mM sodium phosphate, 145 mM sodium chloride, 0.05% Tween™ 80, pH 7.4.222. The derivative of any one of embodiments 1-221, wherein bloodglucose is determined, and/or the mice are weighed, at time −½h (half anhour prior to dosing (t=0)), and at times 1, 2, 4, 8, 24, 48, 72, and 96h.223. The derivative of any one of embodiments 1-222, wherein the glucoseconcentration is measured using the glucose oxidase method.224. The derivative of any one of embodiments 1-223, wherein

(i) ED₅₀ (body weight (BW)) is calculated as the dose giving rise tohalf-maximum effect on delta (e.g., decrease) BW 24 hours following thesubcutaneous administration of the derivative; and/or

(ii) ED₅₀ (blood glucose (BG)) is calcualated as the dose giving rise tohalf-maximum effect on AUC (Area Under the Curve) delta (e.g., decrease)BG 8 hours following the subcutaneous administration of the derivative.

225. The derivative of any one of embodiments 1-224, wherein a sigmoidaldose-response relationship exists, preferably with a clear definition ofthe maximum response.

226. The derivative of any one of embodiments 1-225, which has a moreprotracted profile of action than liraglutide.

227. The derivative of embodiment 226, wherein protraction meanshalf-life in vivo in a relevant animal species, such as db/db mice, rat,pig, and/or, preferably, minipig; wherein the derivative is administeredi) s.c., and/or, preferably, ii) s.c.

228. The derivative of any one of embodiments 1-227, wherein theterminal half-life (T_(1/2)) after i.v. administration in minipigs is

a) at least 12 hours, preferably at least 24 hours, more preferably atleast 36 hours, even more preferably at least 48 hours, or mostpreferably at least 60 hours;

b) at least 7 hours, preferably at least 16 hours, more preferably atleast 24 hours, even more preferably at least 30 hours, or mostpreferably at least 40 hours;

c) at least 44 hours, preferably at least 55 hours, more preferably atleast 66 hours, even more preferably at least 77 hours, or mostpreferably at least 88 hours; or

d) at least 0.2 times the half-life of semaglutide, preferably at least0.4 times the half-life of semaglutide, more preferably at least 0.6times the half-life of semaglutide, even more preferably at least 0.8times the half-life of semaglutide, or most preferably at least the sameas the half-life of semaglutide.229. The derivative of embodiment 228, wherein the minipigs are maleGöttingen minipigs.230. The derivative of any one of embodiments 227-229, wherein theminipigs are 7-14 months of age, and preferably weighing from 16-35 kg.231. The derivative of any one of embodiments 227-230, wherein theminipigs are housed individually, and fed once or twice daily,preferably with SDS minipig diet.232. The derivative of any one of embodiments 227-231, wherein thederivative is dosed, i.v., after at least 2 weeks of acclimatisation.233. The derivative of any one of embodiments 227-232, wherein theanimals are fasted for approximately 18 h before dosing and for at least4 h after dosing, and have ad libitum access to water during the wholeperiod.234. The derivative of any one of embodiments 227-233, wherein the GLP-1derivative is dissolved in 50 mM sodium phosphate, 145 mM sodiumchloride, 0.05% tween 80, pH 7.4 to a suitable concentration, preferablyfrom 20-60 nmol/ml.235. The derivative of any one of embodiments 227-234, whereinintravenous injections of the derivative are given in a volumecorresponding to 1-2 nmol/kg.236. The derivative of any one of embodiments 1-235, which increases theglucose stimulated insulin secretion in minipigs.237. The derivative of embodiment 236, wherein the minipigs are maleGöttingen minipigs.238. The derivative of any one of embodiments 236-237, wherein theminipigs are 7-14 months of age.239. The derivative of any one of embodiments 236-238, wherein theminipigs are housed in single pens, and fed once or twice daily,preferably with SDS minipig fodder.240. The derivative of any one of embodiments 236-239, wherein a singledose, optionally after a period with dose escalation, is given i.v., ors.c., in the thin skin behind the ear.2-[1. The derivative of any one of embodiments 236-240, wherein theanimals are fasted for approximately 18 h before dosing.242. The derivative of any one of embodiments 236-2-[1, wherein abaseline group and a number of derivative dose groups corresponding to2-6 different plasma concentration levels are tested, wherein thebaseline group is a) vehicle treated, or b) untreated.243. The derivative of any one of embodiments 236-242, wherein theplasma concenctration level is 3000-80000 pM.244. The derivative of any one of embodiments 236-243, wherein a 1 or 2hour intravenous glucose tolerance test (IVGTT) is performed.245. The derivative of any one of embodiments 236-244, wherein 0.3 g/kgglucose is given i.v. over a period of 30 seconds, and blood samplestaken at suitable time points, such as the following time points (t=0corresopnds to the glucose bolus): −10, −5,0,2, 5, 10, 15, 20, 25, 30,40, 50, 60, 70, 80, 90, 100, 110, 120 minutes.246. The derivative of any one of embodiments 236-245, wherein theconcentration in plasma of the derivative, glucose, and insulin isdetermined.247. The derivative of any one of embodiments 236-246, wherein thederivative concentration is measured at t=0 min, and, optionally, at theend of the test (t=60 min, or t=120 min).248. The derivative of any one of embodiments 236-247, wherein glucoseis analyzed using the glucose oxidase method.249. The derivative of any one of embodiments 236-248, wherein the areaunder the insulin curve (AUCinsulin) is calculated and used as a measureof insulin secretion.250. The derivative of any one of embodiments 236-249, wherein for atleast one concentration thereof, the AUCinsulin is higher than thebaseline AUCinsulin, preferably at least 110% thereof, more preferablyat least 120% thereof, even more preferably at least 130% thereof ormost preferably at least 140% thereof.251. The derivative of any one of embodiments 1-250, which causes areduced feed intake in pigs relative to a control (preferablyvehicle-treated, or untreated);

optionally the feed intake (0-24 h) may be 90% or lower relative to thevehicle-treated control, preferably 80% or lower, more preferably 70% orlower, even more preferably 60% or lower, or most preferably 50% orlower;

wherein feed intake (0-24 h) refers to the first 24 hours afteradministration of the derivative or vehicle.

252. The derivative of embodiment 251, wherein the pigs are femaleLandrace Yorkshire Duroc (LYD) pigs.

253. The derivative of any one of embodiments 251-252, wherein the pigsare 3 months of age, and preferably have a weight of 30-35 kg.

254. The derivative of any one of embodiments 251-253, where the animalsare housed in a group for 1-2 weeks for acclimatisation.

255. The derivative of any one of embodiments 251-254, wherein duringthe experimental period the animals are placed in individual pens fromMonday morning to Friday afternoon for measurement of individual foodintake.

256. The derivative of any one of embodiments 251-255, wherein theanimals are fed ad libitum with pig fodder (such as Svinefoder,Antonio).

257. The derivative of any one of embodiments 251-256, wherein foodintake is monitored on line by logging the weight of fodder every 15minutes, preferably using the Mpigwin system.

258. The derivative of any one of embodiments 251-257, which is dosed0.3, 1.0, 3.0, 10, or 30 nmol/kg, preferably dissolved in a phosphatebuffer (50 mM phosphate, 0.05% Tween™ 80, pH 8), more preferably atconcentrations of 12, 40, 120, 400, or 1200 nmol/ml.259. The derivative of any one of embodiments 251-258, wherein thephosphate buffer serves as vehicle.260. The derivative of any one of embodiments 251-259, wherein theanimals are dosed with a single subcutaneous dose of the derivative, orvehicle (preferably with a dose volume of 0.025 ml/kg), on the morningof day 1, and food intake is measured for 4 days after dosing.261. The derivative of any one of embodiments 1-260, which has an invitro half-life (T_(1/2)) in an extract of rat small intestines, dividedby the corresponding half-life (T_(1/2)) of GLP-1(7-37), of at least0.4, preferably above 0.5, more preferably above 1.0, even morepreferably above 2.0, still more preferably above 3.0, or mostpreferably above 4.0.262. The derivative of any one of embodiments 1-261, which has an invitro half-life (T_(1/2)) in an extract of rat small intestines, dividedby a corresponding half-life (T_(1/2)) of GLP-1(7-37), of above 5.0,preferably above 6.0, more preferably above 7.0, even more preferablyabove 8.0, still more preferably above 9.0, or most preferably above10.0.263. The derivative of any one of embodiments 261-262, wherein the ratsmall intestine extract is prepared as described in Example 57, thederivative is incubated for one hour at 37° C., the concentration of theextract is titrated so that the half-life of GLP-1(7-37) is in the rangeof 10-20 minutes, e.g. 1.4 ug/ml, the resulting samples are analysed byUPLC and/or MALDI-TOF, and/or the incubation and analysis is performedas described in Example 57.264. The derivative of any one of embodiments 1-263, for which a ratio[half-life (T_(1/2)) in vitro in rat small intestine extract, divided bya half-life (T_(1/2)) in vitro in rat small intestine extract ofGLP-1(7-37)] is at least 0.5 times the corresponding ratio ofsemaglutide, preferably at least 2 times the ratio of semaglutide, morepreferably at least 3 times the ratio of semaglutide, even morepreferably at least 5 times the ratio of semaglutide, or most preferablyat least 7 times the ratio of semaglutide.265. The derivative of any one of embodiments 1-264, for which a ratio[half-life (T_(1/2)) in rat small intestine extract, divided by ahalf-life (T_(1/2)) in rat small intestine extract of GLP-1(7-37)] is atleast 0.1 times the corresponding ratio of liraglutide, preferably atleast 0.4 times the ratio of liraglutide, more preferably at least 0.8times the ratio of liraglutide, even more preferably at least 1.2 timesthe ratio of liraglutide, or most preferably at least 1.5 times theratio of liraglutide.266. The derivative of any one of embodiments 1-265, which has ahalf-life (T_(1/2)) in vivo in rats after i.v. administration of atleast 4 hours, preferably at least 6 hours, even more preferably atleast 8 hours, or most preferably at least 10 hours.267. The derivative of any one of embodiments 1-266, which has ahalf-life (T_(1/2)) in vivo in rats after i.v. administration of atleast 12 hours, preferably at least 15 hours, even more preferably atleast 18 hours, or most preferably at least 20 hours.268. The derivative of any one of embodiments 1-266, which has ahalf-life (T_(1/2)) in vivo in rats after i.v. administration of atleast 24 hours, preferably at least 26 hours, or most preferably atleast 30 hours.269. The derivative of any one of embodiments 266-268, in which the ratsare male Sprague Dawley rats with a body weight from 300 to 600 g.270. The derivative of any one of embodiments 1-269, which has ahalf-life (T_(1/2)) in vivo in rats after i.v. administration which isat least the same as the half-life of semaglutide, preferably at least 2times the half-life of semaglutide, more preferably at least 3 times thehalf-life of semaglutide, even more preferably at least 4 times thehalf-life of semaglutide, or most preferably at least 5 times thehalf-life of semaglutide.271. The derivative of any one of embodiments 1-270, which is not thecompound of Examples 17, 21, 33, 34, 35, and 36; preferably not Chem.36, Chem. 40, Chem. 52, Chem. 53, Chem. 54, and Chem. 55.272. The derivative of any one of embodiments 1-271, which is not thecompound of Examples 22, 23, 27, and 41; preferably not Chem. 41, Chem.42, Chem. 46, and Chem. 60.273. The derivative of Example 19; preferably Chem. 38.274. The derivative of Example 10; preferably Chem. 29.275. An intermediate product in the form of a GLP-1 analogue whichcomprises the following modifications as compared to GLP-1(7-37) (SEQ IDNO: 1): (A) (i) (8Aib, 31H, 34Q, 37K); (ii) (des7-8, 34R, 37K, 38E);(iii) (des7-8, 34R, 37K); (iv) (8Aib, 9G, 34R, 37K); (v) (8Aib, 23R,34R, 37K); (vi) (31H, 34Q, 37K); (vii) (9Q, 34R, 37K); (iix) (30E, 34R,37K); (ix) (34R, 37K, 38G); (x) (34R, 36G, 37K); or (xi) (34R, 37K,38E);

wherein the analogue is preferably selected from the following analoguesof GLP-1(7-37) (SEQ ID NO: 1):

(B) (i-a) (8Aib, 31H, 34Q, 37K); (ii-a) (des7-8, 34R, 37K, 38E); (iii-a)(des7-8, 34R, 37K); (iv-a) (8Aib, 9G, 34R, 37K); (v-a) (8Aib, 23R, 34R,37K); (vi-a) (31H, 34Q, 37K); (vii-a) (9Q, 34R, 37K); (iix-a) (30E, 34R,37K); (ix-a) (34R, 37K, 38G); (x-a) (34R, 36G, 37K); (xi-a) (34R, 37K,38E); (xii-a) (7Imp, 34R, 37K); (xiii-a) (8Aib, 34R, 37K); and (xiv-a)(34R, 37K);or a pharmaceutically acceptable salt, amide, or ester of any of theanalogues of (A) or (B).276. The analogue of embodiment 275, wherein the comparison withGLP-1(7-37) (SEQ ID NO: 1) is made by handwriting and eyeballing.277. The analogue of embodiment 275, wherein the comparison withGLP-1(7-37) (SEQ ID NO: 1) is made by use of a standard protein orpeptide alignment program.278. The analogue of embodiment 277, wherein the alignment program is aNeedleman-Wunsch alignment.279. The analogue of any one of embodiment 277-278, wherein the defaultscoring matrix and the default identity matrix is used.280. The analogue of any one of embodiments 277-279, wherein the scoringmatrix is BLOSUM62.281. The analogue of any one of embodiments 277-280, wherein the penaltyfor the first residue in a gap is −10 (minus ten).282. The analogue of any one of embodiments 277-281, wherein thepenalties for additional residues in a gap is −0.5 (minus point five).283. The analogue of any one of embodiments 277-282, which has GLP-1activity.284. The analogue of embodiment 283, wherein GLP-1 activity is definedas described in embodiments 192-196.285. An intermediate product comprising a protracting moiety selectedfrom Chem. 2c, Chem. 3b, and Chem. 4b:HOOC—C₆H₄—O—(CH₂)_(y)—CO—PG  Chem. 2c:R¹—C₆H₄—(CH₂)_(z)—CO—PG  Chem. 3b:HOOC—C₄H₂—(CH₂)_(w)—CO—PG  Chem. 4b:

in which y is an integer in the range of 3-17, z is an integer in therange of 1-5, R¹ is a group having a molar mass not higher than 150 Da,w is an integer in the range of 6-18, and *—PG is a protection group;wherein, optionally, the distal *—COOH group of the protracting moiety,if present, is also protected; or a pharmaceutically acceptable salt,amide or ester thereof.

286. The intermediate product of embodiment 285, wherein *—CO—PG isi)*—COOH, or ii) an activated ester.

287. The intermediate product of embodiment 286, wherein the activatedester is an ester of p-nitrophenol; 2,4,5-trichlorophenol;N-hydroxysuccinimide; N-hydroxysulfosuccinimide;3,4-dihydro-3-hydroxy-1,2,3-benzotriazine-4-one;5-chloro-8-hydroxyquinoline; N-hydroxy-5-norbornene-2,3-dicarboxylicacid imide; pentafluorophenol; p-sulfotetrafluorophenol;N-hydroxyphthalimide; 1-hydroxybenzotriazole;1-hydroxy-7-azabenzotriazole; N-hydroxymaleimide;4-hydroxy-3-nitrobenzene sulfonic acid; or any other activated esterknown in the art.288. The intermediate product of any one of embodiments 285-287, whichcomprises a) a protracting moiety selected from Chem. 2, Chem. 3, andChem. 4:HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:R¹—C₆H₄—(CH₂)_(z)—CO—*  Chem. 3:HOOC—C₄H₂—(CH₂)_(w)—CO—*  Chem. 4:in which y is an integer in the range of 3-17, z is an integer in therange of 1-5, R¹ is a group having a molar mass not higher than 150 Da,and w is an integer in the range of 6-18; andb) a linker selected from Chem. 5b, Chem. 6, and Chem. 7:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5; and PG is a protection group; wherein, optionally, the*—COOH group of the protracting moiety, if present, is preferably alsoprotected as is known in the art, preferably functionalised as anon-reactive ester; or a pharmaceutically acceptable salt, amide, orester thereof.289. The intermediate product of any one of embodiments 285-288, whereinthe linker is as defined in any one of embodiments 1-57.290. The intermediate product of any one of embodiments 285-289, whereinthe protracting moiety is as defined in any one of embodiments 1-87.291. An intermediate product comprising, preferably consisting of,a) a protracting moiety selected from Chem. 2, Chem. 3, and Chem. 4:HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:R¹—C₆H₄—(CH₂)_(z)—CO—*  Chem. 3:HOOC—C₄H₂—(CH₂)_(w)—CO—*  Chem. 4:

in which y is an integer in the range of 3-17, z is an integer in therange of 1-5, R¹ is a group having a molar mass not higher than 150 Da,and w is an integer in the range of 6-18; and

b) a linker comprising Chem. 5b:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5; and PG is a protection group;wherein, optionally, the distal *—COOH group of the protracting moiety,if any, is also protected as is known in the art; preferably under theformation of a non-reactive ester; more preferably i) an ester of analcohol with a bulky side chain, such as an ester of a phenol,optionally substituted; or ii) an ester of branched alkyl, preferablylower alkyl; most preferably protected as OtBu, OBz, and the like; or apharmaceutically acceptable salt, amide, or ester thereof.292. An intermediate product, preferably according to any one ofembodiments 285-291, selected from the following:

wherein, optionally, one or more of the *—COOH group(s), preferably thedistal *—COOH group of the protracting moiety is also protected.293. A derivative according to any one of embodiments 1-274, for use asa medicament.294. A derivative according to any one of embodiments 1-274, for use inthe treatment and/or prevention of all forms of diabetes and relateddiseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving n-cell function, and/or for delaying or preventing diabeticdisease progression.295. Use of a derivative according to any one of embodiments 1-274 inthe manufacture of a medicament for the treatment and/or prevention ofall forms of diabetes and related diseases, such as eating disorders,cardiovascular diseases, gastrointestinal diseases, diabeticcomplications, critical illness, and/or polycystic ovary syndrome;and/or for improving lipid parameters, improving β-cell function, and/orfor delaying or preventing diabetic disease progression.296. A method for treating or preventing all forms of diabetes andrelated diseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression—by administering a pharmaceutically active amount ofa derivative according to any one of embodiments 1-274.297. A derivative of a GLP-1 analogue, which comprises a protractingmoiety selected from Chem. 2, Chem. 3, and Chem. 4:HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:R¹—C₆H₄—(CH₂)_(z)—CO—*  Chem. 3:HOOC—C₄H₂—(CH₂)_(w)—CO—*  Chem. 4:

in which x is an integer in the range of 6-18, y is an integer in therange of 3-17, z is an integer in the range of 1-5, R¹ is a group havinga molar mass not higher than 150 Da, and w is an integer in the range of6-18;

or a pharmaceutically acceptable salt, amide, or ester thereof.

298. The derivative of embodiment 297, wherein the GLP-1 analogue is asdefined in any one of embodiments 1-296.

299. The derivative of any one of embodiments 297-298, wherein theprotracting moiety is as defined in any one of embodiments 1-296.

300. The derivative of any one of embodiments 297-299, which furthercomprises a linker, preferably as defined in any one of embodiments1-296.

ADDITIONAL PARTICULAR EMBODIMENTS

The following are additional particular embodiments of the invention:

1. A derivative of a GLP-1 analogue, wherein the GLP-1 analogue isK³⁷-GLP-1(7-37) or an analogue thereof having up to six amino acidresidues changed as compared to GLP-1(7-37) (SEQ ID NO: 1), whichderivative has two albumin binding moieties attached to K²⁶ and K³⁷,respectively, wherein the albumin binding moiety comprises a protractingmoiety selected from HOOC—(CH₂)_(n)—CO—, HOOC—C₆H₄—O—(CH₂)_(m)—CO—, andR¹—C₆H₄—(CH₂)_(p)—CO—, in which n is an integer in the range of 8-16, mis an integer in the range of 7-17, p is an integer in the range of 1-5,and R¹ is a group having a molar mass not higher than 150 Da; or apharmaceutically acceptable salt, amide, or ester thereof.2. The derivative of embodiment 1, in which n is an even number.3. The derivative of embodiment 2, in which n is 8, 10, 12, 14, or 16;preferably 10, 12, or 14.4. The derivative of embodiment 1, in which m is an odd number.5. The derivative of embodiment 4, in which m is 7, 9, 11, 13, 15, or17; preferably 9, 11, or 15; most preferably 9.6. The derivative of embodiment 1, in which p is an odd number.7. The derivative of embodiment 6, in which p is 1, 3, or 5, preferably3.8. The derivative of any one of embodiments 1 and 4-5, in which the COOHgroup is in the meta- or para-position, preferably in the para-position.9. The derivative of any one of embodiments 1-8, in which R¹ has a molarmass not higher than 130 Da, preferably not higher than 100 Da, morepreferably not higher than 75 Da, even more preferably not higher than60 Da, or most preferably not higher than 50 Da.10. The derivative of any one of embodiments 1-9, in which R¹ has amolar mass not higher than 40 Da, preferably not higher than 30 Da, morepreferably not higher than 20 Da, or most preferably not higher than 15Da.11. The derivative of any one of embodiments 1-10, wherein R¹ isselected from halogen, and straight-chain or branched alkyl having from1-5 C-atoms.12. The derivative of any one of embodiments 1 and 6-7, in which R¹ ismethyl or tert-butyl.13. The derivative of embodiment 12, in which R¹ is in thepara-position.14. The derivative of any one of embodiments 1 and 6-7, in which R¹ is—I.15. The derivative of embodiment 14, in which R¹ is in thepara-position.16. The derivative of any one of embodiments 1-15, in which the GLP-1analogue has a maximum of five, preferably a maximum of four, morepreferably a maximum of three, or most preferably a maximum of two aminoacid changes, as compared to GLP-1(7-37) (SEQ ID NO: 1).17. The derivative of any one of embodiments 1-16, in which the GLP-1analogue has a C-terminal amide.18. The derivative of any one of embodiments 1-16, in which the GLP-1analogue has a C-terminal —COOH group, or a pharmaceutically acceptablesalt thereof.19. The derivative of any one of embodiments 1-18, in which the GLP-1analogue comprises at least one deletion, as compared to GLP-1(7-37)(SEQ ID NO: 1).20. The derivative of any one of embodiments 1-19, in which one or twoamino acids have been deleted at the N-terminus of the GLP-1 analogue,so that the analogue preferably comprises des7, des8, or (des7+des8);more preferably des7, or (des7+des8).21. The derivative of any one of embodiments 1-20, wherein the GLP-1analogue is an analogue of GLP-1(8-37) or GLP-1(9-37) having up to sixamino acid residues changed as compared to GLP-1(7-37) (SEQ ID NO: 1).22. The derivative of any one of embodiments 1-21, wherein the GLP-1analogue is selected from the following: (i) K³⁷-GLP-1(7-37), (ii)K³⁷-GLP-1(8-37), (iii) K³⁷-GLP-1(9-37), or (iv) an analogue of any oneof (i)-(iii) having up to six amino acid residue changes as compared toGLP-1(7-37) (SEQ ID NO: 1).23. The derivative of any one of embodiments 20-21 or 22(ii)-(iv),wherein a His-mimetic or a His-Ala-mimetic has been added to the newN-terminal amino acid.24. The derivative of any one of embodiments 20-23, wherein a derivativeof an imidazole with a free carboxylic acid group has been covalentlycoupled to the N-terminus, preferably via formation of an amide bondbetween the free carboxylic acid group and the N-terminal amino group.25. The derivative of embodiment 24, wherein the imidazole derivative isa mono-substituted imidazole.26. The derivative of embodiment 25, wherein the imidazole issubstituted with a carboxylic acid radical of a lower alkyl having fromone to six carbon atoms.27. The derivative of embodiment 26, wherein the carboxylic acid radicalis selected from acetyl; and straight or branched propionyl, butyryl,pentanoyl; preferably acetyl.28. The derivative of any one of embodiments 1-27, wherein the aminoacid residue at position 8 of the GLP-1 analogue has3H-Imidazol-4-yl-acetyl attached to its N-atom.29. The derivative of any one of embodiments 1-28, wherein the aminoacid residue at position 8 of the GLP-1 analogue is alanine.30. The derivative of embodiment 25, wherein the imidazole issubstituted with (methylcarbamoyl)-2-methyl-propionyl,(ethylcarbamoyl)-2-methyl-propionyl,(propylcarbamoyl)-2-methyl-propionyl, or(butylcarbamoyl)-2-methyl-propionyl.31. The derivative of embodiment 30, wherein the imidazole issubstituted with (methylcarbamoyl)-2-methyl-propionyl,(ethylcarbamoyl)-2-methyl-propionyl, or(propylcarbamoyl)-2-methyl-propionyl, preferably with(ethylcarbamoyl)-2-methyl-propionyl.32. The derivative of any one of embodiments 1-31, wherein the aminoacid residue at position 9 of the GLP-1 analogue has{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl} attached toits N-atom.33. The derivative of any one of embodiments 1-32, wherein the aminoacid residue at position 9 of the GLP-1 analogue is glutamic acid.34. The derivative of any one of embodiments 1-33, which, in addition to37K, comprises at least one of the following substitutions: 8Aib; 31H;34E,Q,R; and/or 38E.35. The derivative of embodiment 34 which comprises 8Aib.36. The derivative of embodiment 34, which comprises 34E, 34Q, or 34R;preferably 34R.37. The derivative of embodiment 35, which further comprises 34R.38. The derivative of embodiment 34, which comprises 31H.39. The derivative of embodiment 35, which further comprises 31H and/or34Q, preferably both.40. The derivative of embodiment 34, which comprises 34R.41. The derivative of embodiment 34, which comprises 38E.42. The derivative of embodiment 37, which further comprises 38E.43. The derivative of any one of embodiments 1-42 in which the twoalbumin binding moieites are similar; preferably substantiallyidentical; or, most preferably, identical.44. The derivative of any one of embodiments 1-43 in which the twoprotracting moieties are similar; preferably substantially identical;or, most preferably, identical.45. The derivative of any one of embodiments 1-44 in which the twoalbumin binding moieties, and/or the two protracting moieties have apercentage of identity of at least 80%, preferably at least 85%, morepreferably at least 90%, or even more preferably at least 95%, or mostpreferably at least 99%.46. The derivative of embodiment 45, wherein the percentage of identityis determined using datamodelling with the Tanimoto similaritycoefficient and the ECFP_(—)6 extended connectivity fingerprints.47. The derivative of any one of embodiments 1-46, in which the albuminbinding moieties are attached to the epsilon amino group of the lysineresidues at position 26 and 37, respectively, via amide bonds,optionally via a linker moiety.48. The derivative of any one of embodiments 1-47 in which the albuminbinding moiety comprises a linker moiety, which at one end is attached,via an amide bond, to the CO-group of the protracting moiety, and at theother end is attached, via an amide bond, to the epsilon amino group ofthe lysine residues at position 26 and 37, respectively.49. The derivative of any one of embodiments 47-48 in which the linkermoiety has from 5 to 30 C-atoms, preferably from 5 to 25 C-atoms, morepreferably from 5 to 20 C-atoms, or most preferably from 5 to 17C-atoms.50. The derivative of any one of embodiments 47-49 in which the linkermoiety has from 4 to 20 hetero atoms, preferably from 4 to 18 heteroatoms, more preferably from 4 to 14 hetero atoms, or most preferablyfrom 4 to 12 hetero atoms.51. The derivative of embodiment 50 in which the hetero atoms are N—,and/or O-atoms.52. The derivative of any one of embodiments 47-51 in which the linkermoiety is selected from the following:

53. The derivative of any one of embodiments 47-52, in which the linkermoiety comprises at least one OEG radical, and/or at least one Glu(glutamic acid) radical.54. The derivative of embodiment 53, in which the linker consists of oneOEG radical, or one Glu radical, the gamma-carboxylic acid group ofwhich preferably forms an amide bond with the epsilon amino group of thelysine residue.55. The derivative of embodiment 53, in which the linker consists of twoOEG radicals, or two Glu radicals, the radicals being interconnected viaamide bonds, and so that, preferably, in case of two Glu radicals, thegamma-carboxylic acid group of one Glu forms an amide bond with theepsilon amino group of the lysine residue, or—more preferably “and” —thegamma-carboxylic acid group of the other Glu forms an amide bond withthe amino group of the first Glu.56. The derivative of embodiment 53, in which the linker comprises atleast one OEG radical and at least one Glu radical, preferably one ofeach, more preferably with the carboxy end of the OEG radical forming anamide bond with the epsilon amino group of the lysine residue, and theamino end of the OEG radical forming an amide bond with thegamma-carboxy group of the Glu radical.57. The derivative of embodiment 56, in which the linker consists of oneGlu radical and two OEG radicals, preferably selected from thefollowing: -Glu-OEG-OEG-, —OEG-Glu-OEG-, and —OEG-OEG-Glu-, in which theamino group of the leftmost radical forms an amide bond with theprotractor moiety, and the carboxy group of the rightmost radical formsan amide bond with the epsilon amino group of the lysine residue,preferably, in case of a Glu radical at the rightmost end, itsgamma-carboxy group is used for the amide bond.58. The derivative of any one of embodiments 1-57 which has a potency(EC₅₀) at or below 3000 pM, preferably below 3000 pM, more preferablybelow 2500 pM, even more preferably below 2000 pM, or most preferablybelow 1500 pM.59. The derivative of any one of embodiments 1-58 which has a potency(EC₅₀) below 1000 pM, preferably below 800 pM, more preferably below 600pM, even more preferably below 400 pM, or most preferably below 200 pM.60. The derivative of any one of embodiments 1-59 which has a potency(EC₅₀) below 180 pM, preferably below 160 pM, more preferably below 140pM, even more preferably below 120 pM, or most preferably below 100 pM.61. The derivative of any one of embodiments 1-60 which has a potency(EC₅₀) below 80 pM, preferably below 60 pM, more preferably below 50 pM,even more preferably at or below 40 pM, or most preferably below 30 pM.62. The derivative of any one of embodiments 58-61, wherein the potencyis determined as stimulation of the formation of cAMP in a mediumcontaining the human GLP-1 receptor, preferably using a stabletransfected cell-line such as BHK467-12A (tk-ts13), and/or using for thedetermination of cAMP a functional receptor assay, e.g. based oncompetition between endogenously formed cAMP and exogenously addedbiotin-labelled cAMP, in which assay cAMP is more preferably capturedusing a specific antibody, and/or wherein an even more preferred assayis the AlphaScreen cAMP Assay, most preferably the one described inExample 50.63. The derivative of any one of embodiments 1-62, the potency (EC₅₀) ofwhich is less than 10 times the potency of semaglutide, preferably lessthan 8 times the potency of semaglutide, more preferably less than 6times the potency of semaglutide, even more preferably less than 4 timesthe potency of semaglutide, or most preferably less than 2 times thepotency of semaglutide.64. The derivative of any one of embodiments 1-63, the potency (EC₅₀) ofwhich is less than 10 times the potency of liraglutide, preferably lessthan 8 times the potency of liraglutide, more preferably less than 6times the potency of liraglutide, even more preferably less than 4 timesthe potency of liraglutide, or most preferably less than 2 times thepotency of liraglutide.65. The derivative of any one of embodiments 1-64, the potency (EC₅₀) ofwhich is less than the potency of liraglutide, preferably less than 0.8times the potency of liraglutide, more preferably less than 0.6 timesthe potency of liraglutide, even more preferably less than 0.5 times thepotency of liraglutide, or most preferably less than or at 0.4 times thepotency of liraglutide.66. The derivative of any one of embodiments 1-65, for which the ratio[GLP-1 receptor binding affinity (IC₅₀) in the presence of 2.0% humanserum albumin (HSA), divided by GLP-1 receptor binding affinity (IC₅₀)in the presence of 0.005% HSA] is at least 1, preferably at least 10,more preferably at least 20, even more preferably at least 30, or mostpreferably at least 40.67. The derivative of any one of embodiments 1-66, for which the ratio[GLP-1 receptor binding affinity (IC₅₀) in the presence of 2.0% humanserum albumin (HSA), divided by GLP-1 receptor binding affinity (IC₅₀)in the presence of 0.005% HSA] is at least 50, preferably at least 60,more preferably at least 70, even more preferably at least 80, or mostpreferably at least 90.68. The derivative of any one of embodiments 1-67, for which the ratio[GLP-1 receptor binding affinity (IC₅₀) in the presence of 2.0% humanserum albumin (HSA), divided by GLP-1 receptor binding affinity (IC₅₀)in the presence of 0.005% HSA], is at least 100, preferably at least120, more preferably at least 140, still more preferably at least 160,even more preferably at least 180, or most preferably at least 200.69. The derivative of any one of embodiments 1-68, the GLP-1 receptorbinding affinity (IC₅₀) of which is measured by way of its ability todisplace ¹²⁵I-GLP-1 from the receptor, the receptor being preferablyprovided in the form of membranes from a stable cell-line such as BHKtk-ts13 transfected with the human GLP-1 receptor; and/or using a SPAbinding assay, preferably employing SPA-particles such as Wheat germagglutinin SPA beads, the binding assay being most preferably performedas described in Example 51.70. The derivative of any one of embodiments 1-69, for which the ratio[GLP-1 receptor binding affinity (IC₅₀) in the presence of 2.0% humanserum albumin (HSA), divided by GLP-1 receptor binding affinity (IC₅₀)in the presence of 0.005% HSA] is at least 20% of the ratio ofsemaglutide, preferably at least 50% of the ratio of semaglutide, morepreferably at least 75% of the ratio of semaglutide, or most preferablyat least equal to the ratio of semaglutide.71. The derivative of any one of embodiments 1-70, for which the ratio[GLP-1 receptor binding affinity (IC₅₀) in the presence of 2.0% humanserum albumin (HSA), divided by GLP-1 receptor binding affinity (IC₅₀)in the presence of 0.005% HSA] is at least equal to the ratio ofliraglutide, preferably at least twice the ratio of liraglutide, morepreferably at least three times the ratio of liraglutide, even morepreferably at least 4 times the ratio of liraglutide, or most preferablyat least 5 times the ratio of liraglutide.72. The derivative of any one of embodiments 1-71, which has an in vitrohalf-life (TA in an extract of rat small intestines, divided by thecorresponding half-life (T_(1/2)) of GLP-1(7-37), of above 0.5,preferably above 1.0, more preferably above 2.0, even more preferablyabove 3.0, or most preferably above 4.0.73. The derivative of any one of embodiments 1-72, which has an in vitrohalf-life (T_(1/2)) in an extract of rat small intestines, divided by acorresponding half-life (T_(1/2)) of GLP-1(7-37), of above 5.0,preferably above 6.0, more preferably above 7.0, or most preferablyabove 8.0.74. The derivative of any one of embodiments 72-73, wherein the ratsmall intestine extract is prepared as described in Example 57, thederivative is incubated for one hour at 37° C., the concentration of theextract is titrated so that the half-life of GLP-1(7-37) is in the rangeof 10-20 minutes, e.g. 1.4 ug/ml, the resulting samples are analysed byUPLC and/or MALDI-TOF, and/or the incubation and analysis is performedas described in Example 57.

75. The derivative of any one of embodiments 1-74, for which a ratio[half-life (T_(1/2)) in vitro in rat small intestine extract, divided bya half-life (T_(1/2)) in vitro in rat small intestine extract ofGLP-1(7-37)] is at least 0.5 times the corresponding ratio ofsemaglutide, preferably at least 2 times the ratio of semaglutide, morepreferably at least 3 times the ratio of semaglutide, even morepreferably at least 5 times the ratio of semaglutide, or most preferablyat least 7 times the ratio of semaglutide.

76. The derivative of any one of embodiments 1-75, for which a ratio[half-life (T_(1/2)) in rat small intestine extract, divided by ahalf-life (T_(1/2)) in rat small intestine extract of GLP-1(7-37)] is

at least 0.1 times the corresponding ratio of liraglutide, preferably atleast 0.4 times the ratio of liraglutide, more preferably at least 0.8times the ratio of liraglutide, even more preferably at least 1.2 timesthe ratio of liraglutide, or most preferably at least 1.5 times theratio of liraglutide.76. The derivative of any one of embodiments 1-75, which has a half-life(T_(1/2)) in vivo in rats after i.v. administration of at least 4 hours,preferably at least 6 hours, even more preferably at least 8 hours, ormost preferably at least 10 hours.77. The derivative of any one of embodiments 1-76, which has a half-life(T_(1/2)) in vivo in rats after i.v. administration of at least 12hours, preferably at least 15 hours, even more preferably at least 18hours, or most preferably at least 20 hours.78. The derivative of any one of embodiments 76-77, in which the ratsare male Sprague Dawley rats with a body weight from 300 to 600 g.79. The derivative of any one of embodiments 1-78, which has a half-life(T_(1/2)) in vivo in rats after i.v. administration which is at leastthe same as the half-life of semaglutide, preferably at least 2 timesthe half-life of semaglutide, more preferably at least 3 times thehalf-life of semaglutide, even more preferably at least 4 times thehalf-life of semaglutide, or most preferably at least 5 times thehalf-life of semaglutide.80. The derivative of any one of embodiments 1-79 which has a half-life(T_(1/2)) in vivo in minipigs after i.v. administration of at least 12hours, preferably at least 24 hours, more preferably at least 36 hours,even more preferably at least 48 hours, or most preferably at least 60hours.81. The derivative of embodiment 80, in which the minipigs are maleGöttingen minipigs.82. The derivative of any one of embodiments 1-81, which has a half-life(T_(1/2)) in vivo in minipigs after i.v. administration which is atleast 0.2 times the half-life of semaglutide, preferably at least 0.4times the half-life of semaglutide, more preferably at least 0.6 timesthe half-life of semaglutide, even more preferably at least 0.8 timesthe half-life of semaglutide, or most preferably at least the same asthe half-life of semaglutide.83. A GLP-1 derivative selected from the following:

-   (i)    N^(ε26)-[2-(2-{2-[10-(4-Carboxyphenoxy)decanoylamino]ethoxy}ethoxy)acetyl],    N^(ε37)-[2-(2-{2-[10-(4-Carboxyphenoxy)decanoylamino]ethoxy}ethoxy)acetyl]-[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7):

-   (ii)    N^(ε26{)2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7):

-   (iii)    N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],    N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7):

-   (iv)    N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],    N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7):

-   (v)    N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],    N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7):

-   (vi)    N^(ε2)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],    N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    amide (SEQ ID NO: 7):

-   (vii)    N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],    N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    amide (SEQ ID NO: 7):

-   (iix)    N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],    N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    amide (SEQ ID NO: 7):

-   (ix)    N^(ε26)-[2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]ethoxy}ethoxy)acetyl],    N^(ε37)-[2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    amide (SEQ ID NO: 7):

-   (x)    N^(ε26)-[2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylaminol    N^(ε37)-[2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]][Aib⁸,Arg³⁴,Lys³⁷]GLP-1    (7-37)-peptide amide (SEQ ID NO: 7):

-   (xi)    N^(ε26)-(2-{2-[2-(2-{2-[2-(13-Carboxy-tridecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl),    N^(ε37)-(2-{2-[2-(2-{2-[2-(13-Carboxy-tridecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl)[Aib⁸,Arg³⁴,Lys³⁷GLP-1(7-37)-peptide    amide (SEQ ID NO: 7):

-   (xii)    N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[4-(4-iodo-phenyl)-butyrylamino]-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[4-(4-iodo-phenyl]-butyrylamino]-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl}    [Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide (SEQ ID NO: 7):

-   (xiii)    AV²⁶-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[16-(4-carboxyphenoxy)hexadecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[16-(4-carboxyphenoxy)hexadecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}-[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7):

-   (xiv)    N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[16-(3-carboxyphenoxy)hexadecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[16-(3-carboxyphenoxy)hexadecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}-[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7):

-   (xv)    N^(ε26)-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)-ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]-butyrylamino}ethoxy)ethoxy]acetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7):

-   (xvi)    N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[12-(3-carboxyphenoxy)dodecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]a    cetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[12-(3-carboxyphenoxy)dodecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]a    cetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide (SEQ ID NO: 7):

-   (xvii)    N^(ε26)-[2-(2-[2-(2-[2-(2-[4-(10-(4-Carboxyphenoxy)decanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]-N^(ε37)-[2-(2-[2-(2-[2-(2-[4-(10-(4-Carboxyphenoxy)decanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)    ethoxy]ethoxy)acetyl][Aib⁸,His³¹,Gln³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 3):

-   (iixx)    N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[4-(4-methylphenyl)butyrylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[4-(4-methylphenyl)butyrylamino]butyrylamino}ethoxy)ethoxy]-acetylamino}ethoxy)ethoxy]acetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7):

-   (ixx)    N^(ε26)-((S)-4-Carboxy-4-{(S)-4-carboxy-4-[10-(4-carboxyphenoxy)-decanoylamino]butyrylamino}butyryl),    N^(ε37)-((S)-4-Carboxy-4-{(S)-4-carboxy-4-[10-(4-carboxyphenoxy)-decanoylamino]butyrylamino}butyryl)[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7):

-   (xx)    N^(ε26)-{2-[2-(2-{2-[2-(2-{4-Carboxy-4-[10-(3-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{4-Carboxy-4-[10-(3-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7):

-   (xxi)    N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],    N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,His³¹,Gln³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 3):

-   (xxii)    N⁹-{2-[2-(1H-Imidazol-4-yl)ethylcarbamoyl]-2-methylpropionyl},    N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}    [Arg³⁴, Lys³¹GLP-1(9-37)Glu³⁸-peptide (SEQ ID NO: 2):

-   (xxiii)    N⁹-{2-[2-(1H-Imidazol-4-yl)ethylcarbamoyl]-2-methylpropionyl]-N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}    [Arg³⁴, Lys³⁷]GLP-1(9-37)-peptide (SEQ ID NO: 11):

-   (xxiv)    N^(ε26)-{2-[2-(2-{(S)-4-Carboxy-4-[2-(2-{2-[(13-carboxytridecanoylamino)]ethoxy}ethoxy)acetylamino]butyrylamino}ethoxy)ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{(S)-4-Carboxy-4-[2-(2-{2-[(13-carboxytridecanoylamino)]ethoxy}ethoxy)acetylamino]butyrylamino}ethoxy)ethoxy]acetyl}-[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7):

-   (xxv)    N^(ε2)-[(S)-4-Carboxy-4-{2-[2-(2-[2-(2-{2-[(13-carboxytridecanoylamino)]ethoxy}ethoxy)acetylamino]ethoxy)ethoxy]acetylamino}butyryl],    N^(ε37)-[(S)-4-Carboxy-4-{2-[2-(2-[2-(2-{2-[(13-carboxytridecanoylamino)]ethoxy}ethoxy)acetylamino]ethoxy)ethoxy]acetylamino}butyryl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide    (SEQ ID NO: 7):

-   (xxvi)    N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-[4-(4-tert-Butyl-phenyl)-butyrylamino]-4-carboxy-butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-[4-(4-tert-Butyl-phenyl)-butyrylamino]-4-carboxy-butyrylaminoyethoxy}-ethoxy)-acetylamino}-ethoxy)-ethoxy]-acetyl}    [Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide (SEQ ID NO: 7):

and

-   (xxvii)    N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methylpropionyl}-N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-[4-(4-tert-Butylphenyl)butyrylamino]-4-carboxybutyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},    N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-[4-(4-tert-Butylphenyl)butyrylamino]-4-carboxybutyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}[Arg³⁴,    Lys³⁷]GLP-1(9-37)-peptide (SEQ ID NO: 11):

or a pharmaceutically acceptable salt, amide, or ester of any of thederivatives (i)-(xxvii).84. A derivative according to any one of embodiments 1-83 for use as amedicament.85. A derivative according to any one of embodiments 1-83, for use inthe treatment and/or prevention of all forms of diabetes and relateddiseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression.86. Use of a derivative according to any one of embodiments 1-83, in themanufacture of a medicament for the treatment and/or prevention of allforms of diabetes and related diseases, such as eating disorders,cardiovascular diseases, gastrointestinal diseases, diabeticcomplications, critical illness, and/or polycystic ovary syndrome;and/or for improving lipid parameters, improving β-cell function, and/orfor delaying or preventing diabetic disease progression.87. A method of treating or preventing all forms of diabetes and relateddiseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression, by administering a pharmaceutically active amountof a derivative according to any one of embodiments 1-83.

EXAMPLES

This experimental part starts with a list of abbreviations, and isfollowed by a section including general methods for synthesising andcharacterising analogues and derivatives of the invention. Then followsa number of examples which relate to the preparation of specific GLP-1derivatives, and at the end a number of examples have been includedrelating to the activity and properties of these analogues andderivatives (section headed pharmacological methods).

The examples serve to illustrate the invention.

Abbreviations

The following abbreviations are used in the following, in alphabeticalorder:

-   Aib: aminoisobutyric acid (α-aminoisobutyric acid)-   API: Active Pharmaceutical Ingredient-   AUC: Area Under the Curve-   BG: Blood Glucose-   BHK Baby Hamster Kidney-   BW: Body Weight-   Bom: benzyloxymethyl-   Boc: t-butyloxycarbonyl-   BSA: Bovine serum albumin-   Bzl: benzyl-   Clt: 2-chlorotrityl-   collidine: 2,4,6-trimethylpyridine-   DCM: dichloromethane-   Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-   DIC: diisopropylcarbodiimide-   DIPEA: diisopropylethylamine-   DMAP: 4-dimethylaminopyridine-   DMEM: Dulbecco's Modified Eagle's Medium (DMEM)-   EDTA: ethylenediaminetetraacetic acid-   EGTA: ethylene glycol tetraacetic acid-   FCS: Fetal Calf Serum-   Fmoc: 9-fluorenylmethyloxycarbonyl-   HATU: (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluoro-phosphate)-   HBTU: (2-(1H-benzotriazol-1-yl-)-1,1,3,3 tetramethyluronium    hexafluorophosphate)-   HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-   HFIP 1,1,1,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol-   HOAt: 1-hydroxy-7-azabenzotriazole-   HOBt: 1-hydroxybenzotriazole-   HPLC: High Performance Liquid Chromatography-   HSA: Human Serum Albumin-   IBMX: 3-isobutyl-1-methylxanthine-   Imp: Imidazopropionic acid (also referred to as des-amino histidine,    DesH)-   i.v. intravenously-   ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl-   IVGTT: Intravenous Glucose Tolerance Test-   LCMS: Liquid Chromatography Mass Spectroscopy-   LYD: Landrace Yorkshire Duroc-   MALDI-MS: See MALDI-TOF MS-   MALDI-TOF MS: Matrix-Assisted Laser Desorption/lonisation Time of    Flight Mass Spectro-scopy-   MeOH: methanol-   Mmt: 4-methoxytrityl-   Mtt: 4-methyltrityl-   NMP: N-methylpyrrolidone-   OBz: benzoyl ester-   OEG: 8-amino-3,6-dioxaoctanic acid-   OPfp: pentafluorophenoxy-   OPnp: para-nitrophenoxy-   OSu: O-succinimidyl esters (hydroxysuccinimide esters)-   OSuc: 2,5-dioxo-pyrrolidin-1-yl-   OtBu: tert butyl ester-   Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-   PBS: Phosphate Buffered Saline-   PD: Pharmacodynamic-   Pen/Strep: Pencillin/Streptomycin-   PK: Pharmacokinetic-   RP: Reverse Phase-   RP-HPLC: Reverse Phase High Performance Liquid Chromatography-   RT: Room Temperature-   Rt: Retention time-   s.c.: Subcutaneously-   SD: Standard Deviation-   SEC-HPLC: Size Exclusion High Performance Liquic Chromatography-   SEM: Standard Error of Mean-   SPA: Scintillation Proximity Assay-   SPPS: Solid Phase Peptide Synthesis-   tBu: tert. butyl-   TFA: trifluoroacetic acid-   TIS: triisopropylsilane-   TLC: Thin Layer Chromatography-   Tos: tosylate (or pare-toluenesulfonyl)-   Tris: tris(hydroxymethyl)aminomethane or    2-amino-2-hydroxymethyl-propane-1,3-diol-   Trt: triphenylmethyl or trityl-   Trx: tranexamic acid-   UPLC: Ultra Performance Liquid Chromatography    Methods of Preparation

A. General Methods

This section relates to methods for solid phase peptide synthesis (SPPSmethods, including methods for de-protection of amino acids, methods forcleaving the peptide from the resin, and for its purification), as wellas methods for detecting and characterising the resulting peptide (LCMS,MALDI, and UPLC methods). The solid phase synthesis of peptides may insome cases be improved by the use of di-peptides protected on thedi-peptide amide bond with a group that can be cleaved under acidicconditions such as, but not limited to, 2-Fmoc-oxy-4-methoxybenzyl, or2,4,6-trimethoxybenzyl. In cases where a serine or a threonine ispresent in the peptide, pseudoproline di-peptides may be used (availablefrom, e.g., Novabiochem, see also W. R. Sampson (1999), J. Pep. Sci. 5,403). The protected amino acid derivatives used were standard Fmoc-aminoacids (supplied from e.g. Anaspec, IRIS, or Novabiochem). The N-terminalamino acid was Boc protected at the alpha amino group (e.g.Boc-His(Boc)-OH, or Boc-His(Trt)-OH for peptides with H is at theN-terminus). The epsilon amino group of lysines in the sequence wereeither protected with Mtt, Mmt, Dde, ivDde, or Boc, depending on theroute for attachment of the albumin binding moiety and spacer. Thealbumin binding moiety and/or linker can be attached to the peptideeither by acylation of the resin bound peptide or by acylation insolution of the unprotected peptide. In case of attachment of thealbumin binding moiety and/or linker to the protected peptidyl resin,the attachment can be modular using SPPS and suitably protected buildingblocks such as but not limited to Fmoc-Oeg-OH(Fmoc-8-amino-3,6-dioxaoctanoic acid), Fmoc-Trx-OH (Fmoc-tranexamicacid), Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester,nonadecanedioic acid mono-tert-butyl ester, or4-(9-carboxynonyloxy)benzoic acid tert-butyl ester.

1. Synthesis of Resin Bound Peptide

SPPS Method A

SPPS method A refers to the synthesis of a protected peptidyl resinusing Fmoc chemistry on an Applied Biosystems 433 peptide synthesiser(also designated AB1433A synthesiser) in 0.25 mmol or 1.0 mmol scaleusing the manufacturers FastMoc UV protocols which employ HBTU or HATUmediated couplings in NMP, and UV monitoring of the de-protection of theFmoc protection group.

The starting resin used for the synthesis of peptide amides was asuitable Rink-Amide resin (for peptide amides), or (for peptides with acarboxy C-terminus) either a suitable Wang resin or a suitablechlorotrityl resin. Suitable resins are commercially available from,e.g., Novabiochem.

SPPS Method B

SPPS method B refers to the synthesis of a protected peptidyl resinusing Fmoc chemistry on a microwave-based Liberty peptide synthesiser(CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-loadWang resin available from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wangresin, 0.35 mmol/g). Fmoc-deprotection was with 5% piperidine in NMP atup to 70 or 75° C. The coupling chemistry was DIC/HOAt in NMP. Aminoacid/HOAt solutions (0.3 M in NMP at a molar excess of 3-10 fold) wereadded to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAtsolution were used per coupling for the following scale reactions:Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling timesand temperatures were generally 5 minutes at up to 70 or 75° C. Longercoupling times were used for larger scale reactions, for example 10 min.Histidine amino acids were double coupled at 50° C., or quadruplecoupled if the previous amino acid was sterically hindered (e.g. Aib).Arginine amino acids were coupled at RT for 25 min then heated to 70 or75° C. for 5 min. Some amino acids such as but not limited to Aib, were“double coupled”, meaning that after the first coupling (e.g. 5 min at75° C.), the resin is drained and more reagents are added (amino acid,HOAt and DIC), and the mixture in heated again (e.g. 5 min at 75° C.).When a chemical modification of a lysine side chain was desired, thelysine was incorporated as Lys(Mtt). The Mtt group was removed bywashing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM andNMP. The chemical modification of the lysine was performed either bymanual synthesis (see SPPS method D) or by one or more automated stepson the Liberty peptide synthesiser as described above, using suitablyprotected building blocks (see General methods), optionally including amanual coupling.

SPPS Method D

SPPS method D refers to synthesis of the protected peptidyl resin usingmanual Fmoc chemistry. This was typically used for the attachment of thelinkers and side chains to the peptide backbone. The followingconditions were employed at 0.25 mmol synthesis scale. The couplingchemistry was DIC/HOAt/collidine in NMP at a 4-10 fold molar excess.Coupling conditions were 1-6 h at room temperature. Fmoc-deprotectionwas performed with 20-25% piperidine in NMP (3×20 ml, each 10 min)followed by NMP washings (4×20 mL). Dde- or ivDde-deprotection wasperformed with 2% hydrazine in NMP (2×20 ml, each 10 min) followed byNMP washings (4×20 ml). Mtt- or Mmt-deprotection was performed with 2%TFA and 2-3% TIS in DCM (5×20 ml, each 10 min) followed by DCM (2×20ml), 10% MeOH and 5% DIPEA in DCM (2×20 ml) and NMP (4×20 ml) washings,or by treatment with neat hexafluoroisopropanol (5×20 ml, each 10 min)followed by washings as above. The albumin binding moiety and/or linkercan be attached to the peptide either by acylation of the resin boundpeptide or acylation in solution of the unprotected peptide (see theroutes described below). In case of attachment of the albumin bindingmoiety and/or linker to the protected peptidyl resin the attachment canbe modular using SPPS and suitably protected building blocks (seeGeneral methods).

Attachment to Resin Bound Peptide—Route I:

Activated (active ester or symmetric anhydride) albumin binding moietyor linker such as octadecanedioic acid mono-(2,5-dioxo-pyrrolidin-1-yl)ester (Ebashi et al. EP511600, 4 molar equivalents relative to resinbound peptide) was dissolved in NMP (25 mL), added to the resin andshaken overnight at room temperature. The reaction mixture was filteredand the resin was washed extensively with NMP, DCM, 2-propanol, methanoland diethyl ether.

Attachment to Resin Bound Peptide—Route II:

The albumin binding moiety was dissolved in NMP/DCM (1:1, 10 ml). Theactivating reagent such as HOBt (4 molar equivalents relative to resin)and DIC (4 molar equivalents relative to resin) was added and thesolution was stirred for 15 min. The solution was added to the resin andDIPEA (4 molar equivalents relative to resin) was added. The resin wasshaken 2 to 24 hours at room temperature. The resin was washed with NMP(2×20 ml), NMP/DCM (1:1, 2×20 ml) and DCM (2×20 ml).

Attachment to Peptide in Solution—Route III:

Activated (active ester or symmetric anhydride) albumin binding moietyor linker such as octadecanedioic acid mono-(2,5-dioxo-pyrrolidin-1-yl)ester (Ebashi et al. EP511600) 1-1.5 molar equivalents relative to thepeptide was dissolved in an organic solvent such as acetonitrile, THF,DMF, DMSO or in a mixture of water/organic solvent (1-2 ml) and added toa solution of the peptide in water (10-20 ml) together with 10 molarequivalents of DIPEA. In case of protecting groups on the albuminbinding residue such as tert-butyl, the reaction mixture was lyophilisedovernight and the isolated crude peptide deprotected afterwards. In caseof tert-butyl protection groups the deprotection was performed bydissolving the peptide in a mixture of trifluoroacetic acid, water andtriisopropylsilane (90:5:5). After for 30 min the mixture was evaporatedin vacuo and the crude peptide purified by preparative HPLC as describedlater.

SPPS Method E

SPPS method E refers to peptide synthesis by Fmoc chemistry on a PreludeSolid Phase Peptide Synthesiser from Protein Technologies (Tucson, Ariz.85714 U.S.A.). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load fmoc-Lys(Mtt)-Wang resin, 0.35mmol/g). Fmoc-deprotection was with 25% piperidine in NMP for 2×10 min.The coupling chemistry was DIC/HOAt/collidine in NMP. Amino acid/HOAtsolutions (0.3 M in NMP at a molar excess of 3-10 fold) were added tothe resin followed by the same molar equivalent of DIC (3 M in NMP) andcollidine (3 M in NMP). For example, the following amounts of 0.3M aminoacid/HOAt solution were used per coupling for the following scalereactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml. Coupling timeswere generally 60 minutes. Some amino acids including, but not limitedto arginine, Aib or histidine were “double coupled”, meaning that afterthe first coupling (e.g. 60 min), the resin is drained and more reagentsare added (amino acid, HOAt, DIC, and collidine), and the mixtureallowed to react gain (e.g. 60 min). Some amino acids and fatty acidderivatives including but not limited to Fmoc-Oeg-OH, Fmoc-Trx-OH,Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester,nonadecanedioic acid mono-tert-butyl ester, or4-(9-carboxynonyloxy)benzoic acid tert-butyl ester were coupled forprolonged time, for example 6 hours. When a chemical modification of alysine side chain was desired, the lysine was incorporated as Lys(Mtt).The Mtt group was removed by washing the resin with DCM and suspendingthe resin in hexafluoroisopropanol/DCM (75:25) for 3×10 minutes followedby washings with DCM, 20% piperidine and NMP. The chemical modificationof the lysine was performed either by manual synthesis (see SPPS methodD) or by one or more automated steps on the Prelude peptide synthesiseras described above using suitably protected building blocks (see Generalmethods).

2. Cleavage of Peptide from the Resin and Purification

After synthesis the resin was washed with DCM, and the peptide wascleaved from the resin by a 2-3 hour treatment with TFA/TIS/water(95/2.5/2.5 or 92.5/5/2.5) followed by precipitation with diethylether.The peptide was dissolved in a suitable solvent (such as, e.g., 30%acetic acid) and purified by standard RP-HPLC on a C18, 5 μM column,using acetonitrile/water/TFA. The fractions were analysed by acombination of UPLC, MALDI and LCMS methods, and the appropriatefractions were pooled and lyophilised.

3. Methods for Detection and Characterisation

LCMS Methods

LCMS Method 1 (LCMS1)

An Agilent Technologies LC/MSD TOF (G1969A) mass spectrometer was usedto identify the mass of the sample after elution from an Agilent 1200series HPLC system. The de-convolution of the protein spectra wascalculated with Agilent's protein confirmation software.

Eluents:

A: 0.1% Trifluoro acetic acid in water

B: 0.1% Trifluoro acetic acid in acetonitrile

Column: Zorbax 5u, 300SB—C3, 4.8×50 mm

Gradient: 25%-95% acetonitrile over 15 min

LCMS Method 2 (LCMS2)

A Perkin Elmer Sciex API 3000 mass spectrometer was used to identify themass of the sample after elution from a Perkin Elmer Series 200 HPLCsystem.

Eluents:

A: 0.05% Trifluoro acetic acid in water

B: 0.05% Trifluoro acetic acid in acetonitrile

Column: Waters Xterra MS C-18×3 mm id 5 pm

Gradient: 5%-90% acetonitrile over 7.5 min at 1.5 ml/min

LCMS Method 3 (LCMS3)

A Waters Micromass ZQ mass spectrometer was used to identify the mass ofthe sample after elution from a Waters Alliance HT HPLC system.

Eluents:

A: 0.1% Trifluoro acetic acid in water

B: 0.1% Trifluoro acetic acid in acetonitrile

Column: Phenomenex, Jupiter C4 50 X 4.60 mm id 5 pm

Gradient: 10%-90% B over 7.5 min at 1.0 ml/min

LCMS Method 4 (LCMS4)

LCMS4 was performed on a setup consisting of Waters Acquity UPLC systemand LCT Premier XE mass spectrometer from Micromass. The UPLC pump wasconnected to two eluent reservoirs containing:

A: 0.1% Formic acid in water

B: 0.1% Formic acid in acetonitrile

The analysis was performed at RT by injecting an appropriate volume ofthe sample (preferably 2-10 μl) onto the column which was eluted with agradient of A and B. The UPLC conditions, detector settings and massspectrometer settings were:

Column: Waters Acquity UPLC BEH, C-18, 1.7 pm, 2.1 mm×50 mm

Gradient: Linear 5%-95% acetonitrile during 4.0 min (alternatively 8.0min) at 0.4 ml/min

Detection: 214 nm (analogue output from TUV (Tunable UV detector))

MS ionisation mode: API-ES

Scan: 100-2000 amu (alternatively 500-2000 amu), step 0.1 amu

UPLC and HPLC methods

Method 05_B5_(—)1

UPLC (method 05_B5_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C. The UPLC system was connected to twoeluent reservoirs containing:

A: 0.2 M Na₂SO₄, 0.04 M H₃PO₄, 10% CH₃CN (pH 3.5)

B: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 60% A, 40% B to 30% A, 70% Bover 8 minutes at a flow-rate of 0.40 ml/min.

Method 05_B7_(—)1

UPLC (method 05_B7_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C. The UPLC system was connected to twoeluent reservoirs containing:

A: 0.2 M Na₂SO₄, 0.04 M H₃PO₄, 10% CH₃CN (pH 3.5)

B: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 80% A, 20% B to 40% A, 60% Bover 8 minutes at a flow-rate of 0.40 ml/min.

Method 04_A2_(—)1

UPLC (method 04_A2_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C. The UPLC system was connected to twoeluent reservoirs containing:

A: 90% H₂O, 10% CH₃CN, 0.25 M ammonium bicarbonate

B: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 90% A, 10% B to 60% A, 40% Bover 16 minutes at a flow-rate of 0.40 ml/min.

Method 04_A3_(—)1

UPLC (method 04_A3_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C. The UPLC system was connected to twoeluent reservoirs containing:

A: 90% H₂O, 10% CH₃CN, 0.25 M ammonium bicarbonate

B: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 75% A, 25% B to 45% A, 55% Bover 16 minutes at a flow-rate of 0.40 ml/min.

Method 04_A4_(—)1

UPLC (method 04_A4_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C. The UPLC system was connected to twoeluent reservoirs containing:

A: 90% H₂O, 10% CH₃CN, 0.25 M ammonium bicarbonate

B: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 65% A, 35% B to 25% A, 65% Bover 16 minutes at a flow-rate of 0.40 ml/min.

Method 08_B2_(—)1

UPLC (method 08_B2_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C. The UPLC system was connected to twoeluent reservoirs containing:

A: 99.95% H₂O, 0.05% TFA

B: 99.95% CH₃CN, 0.05% TFA

The following linear gradient was used: 95% A, 5% B to 40% A, 60% B over16 minutes at a flow-rate of 0.40 ml/min.

Method 08_B4_(—)1

UPLC (method 08_B4_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C. The UPLC system was connected to twoeluent reservoirs containing:

A: 99.95% H₂O, 0.05% TFA

B: 99.95% CH₃CN, 0.05% TFA

The following linear gradient was used: 95% A, 5% B to 95% A, 5% B over16 minutes at a flow-rate of 0.40 ml/min.

Method 05_B10_(—)1

UPLC (Method 05_B10_(—)1): The RP-analyses was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C. The UPLC system was connected to twoeluent reservoirs containing:

A: 0.2 M Na₂SO₄, 0.04 M H₃PO₄, 10% CH₃CN (pH 3.5)

B: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 40% A, 60% B to 20% A, 80% Bover 8 minutes at a flow-rate of 0.40 ml/min.

Method 01_A4_(—)2

UPLC (Method 01_A4_(—)2): The RP-analysis was performed using a Waters600S system fitted with a waters 996 diode array detector. UV detectionsat 214 nm and 254 nm were collected using a Symmetry300 C18, 5 um, 3.9mm×150 mm column, 42° C. The HPLC system was connected to three eluentreservoirs containing: A: 100% H₂O, B: 100% CH₃CN, C: 1% trifluoroaceticacid in H₂O. The following linear gradient was used: 90% A, 5% B, 5% Cto 0% A, 95% B, 5% C over 15 minutes at a flow-rate of 1.0 ml/min.

Method 09_B2_(—)1

UPLC (Method 09_B2_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130A, 1.7um, 2.1 mm×150 mm column, 40° C. The UPLC system was connected to twoeluent reservoirs containing: A: 99.95% H₂O, 0.05% TFA; B: 99.95% CH₃CN,0.05% TFA. The following linear gradient was used: 95% A, 5% B to 40% A,60% B over 16 minutes at a flow-rate of 0.40 ml/min.

Method 09_B4_(—)1

UPLC (Method 09_B4_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C. The UPLC system was connected to twoeluent reservoirs containing: A: 99.95% H₂O, 0.05% TFA; B: 99.95% CH₃CN,0.05% TFA. The following linear gradient was used: 95% A, 5% B to 5% A,95% B over 16 minutes at a flow-rate of 0.40 ml/min.

Method 05_B8_(—)1

UPLC (Method 05_B8_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C. The UPLC system was connected to twoeluent reservoirs containing: A: 0.2 M Na₂SO₄, 0.04 M H₃PO₄, 10% CH₃CN(pH 3.5); B: 70% CH₃CN, 30% H₂O. The following linear gradient was used:50% A, 50% B to 20% A, 80% B over 8 minutes at a flow-rate of 0.40ml/min.

Method 10_B14_(—)1

UPLC (Method 10_B14_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH ShieldRP18, 1.7 um,2.1 mm×150 mm column, 50° C. The UPLC system was connected to two eluentreservoirs containing: A: 99.95% H₂O, 0.05% TFA; B: 99.95% CH₃CN, 0.05%TFA. The following linear gradient was used: 70% A, 30% B to 40% A, 60%B over 12 minutes at a flow-rate of 0.40 ml/min.

Method 04_A6_(—)1

UPLC (Method 04_A6_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130A, 1.7um, 2.1 mm×150 mm column, 40° C. The UPLC system was connected to twoeluent reservoirs containing: A: 10 mM TRIS, 15 mM ammonium sulphate,80% H₂O, 20%, pH 7.3; B: 80% CH₃CN, 20% H₂O. The following lineargradient was used: 95% A, 5% B to 10% A, 90% B over 16 minutes at aflow-rate of 0.35 ml/min.

Method 01_B4_(—)1

HPLC (Method 01_B4_(—)1): The RP-analysis was performed using a Waters600S system fitted with a Waters 996 diode array detector. UV detectionswere collected using a Waters 3 mm×150 mm 3.5 um C-18 Symmetry column.The column was heated to 42° C. and eluted with a linear gradient of5-95% acetonitrile, 90-0% water, and 5% trifluoroacetic acid (1.0%) inwater over 15 minutes at a flow-rate of 1 ml/min.

MALDI-MS Method

Molecular weights were determined using matrix-assisted laser desorptionand ionisation time-of-flight mass spectroscopy, recorded on a Microflexor Autoflex (Bruker). A matrix of alpha-cyano-4-hydroxy cinnamic acidwas used.

NMR Method

Proton NMR spectra were recorded using a Brucker Avance DPX 300 (300MHz) with tetramethylsilane as an internal standard. Chemical shifts (δ)are given in ppm and splitting patterns are designated as follows: s,singlet; d, doublet; dd, double doublet; dt, double triplet t, triplet,tt, triplet of triplets; q, quartet; quint, quintet; sext, sextet; m,multiplet, and br=broad.

B. Synthesis of Intermediates 1. Synthesis of Mono Esters of FattyDiacids

Overnight reflux of the C12, C14, C16 and C18 diacids withBoc-anhydride, DMAP, and t-butanol in toluene gives predominately thet-butyl mono ester. Obtained is after work-up a mixture of mono acid,diacid and diester. Purification is carried out by washing, short plugsilica filtration and crystallisation.

2. Synthesis of 2-(1-Trityl-1H-imidazol-4-yl)-ethyl amine

Histamine dihydrochloride (20.47 g; 0.111 mol) and triethylamine (48 mL;0.345 mol) in absolute methanol (400 mL) were stirred at roomtemperature for 10 min. Trifluoroacetic acid ethyl ester (14.6 mL; 0.122mol) in methanol (30 mL) was added dropwise over 30 min at 0° C.Reaction mixture was stirred for 3.5 hrs at room temperature and then itwas evaporated to dryness in vacuo. The residue was dissolved indichlormethane (450 mL) and triethylamine (31 mL; 0.222 mol) was added.Then trityl chloride (34.1 g; 0.122 mol) was added piecewise and mixturewas stirred over night at room temperature. Chloroform (400 mL) andwater (600 mL) were poured into reaction mixture. Aqueous layer wasseparated and extracted with chloroform (3×400 mL). The combined organiclayers were dried over anhydrous magnesium sulfate. Solvent was removedand the beige solid was triturated with hexanes (1000 mL). Suspensionwas filtered to yield2,2,2-trifluoro-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-acetamide aswhite solid.

Yield: 45.54 g (91%).

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 8.44 (bs, 1H); 7.43 (s, 1H);7.41-7.33 (m, 9H); 7.19-7.10 (m, 6H); 6.65 (s, 1H); 3.66 (q, J=5.9 Hz,2H); 2.79 (t, J=5.9 Hz, 2H).

The above amide (45.54 g; 0.101 mmol) was dissolved in tetrahydrofuran(1000 mL) and methanol (1200 mL). A solution of sodium hydroxide (20.26g; 0.507 mol) in water (500 mL) was added. Mixture was stirred for 2 hrsat room temperature and then it was concentrated in vacuo. The residuewas separated between chloroform (1200 mL) and water (800 mL). Aqueouslayer was extracted with chloroform (3×400 mL). Organic layers werecombined and dried over anhydrous magnesium sulfate. Evaporation of thesolvent yielded brown oil, which was dried for 3 days in vacuo to givethe title product as beige solid.

Yield: 32.23 g (90%).

Overall yield: 82%.

M.p.: 111-113° C.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.39 (d, J=1.3, 1H); 7.38-7.32(m, 9H); 7.20-7.12 (m, 6H); 6.61 (s, 1H); 3.00 (t, J=6.6 Hz, 2H); 2.70(t, J=6.5 Hz, 2H); 1.93 (bs, 2H).

3. Synthesis of2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid

A mixture of Meldrum's acid (5.52 g, 38.3 mmol), potassium carbonate(26.5 g, 191 mmol) and methyl iodide (7.15 mL, 115 mmol) in acetonitrile(75 mL) was heated at 75° C. in a sealed tube for 7 hrs. The mixture wascooled to room temperature, diluted with dichloromethane (300 mL),filtered and the filtrate evaporated to dryness in vacuo. Ethyl acetate(75 mL), hexanes (75 mL) and water (50 mL) were added and phases wereseparated. The organic layer was washed with 10% aqueous solution ofsodium thiosulfate (50 mL) and water (50 mL); dried over anhydrousmagnesium sulfate and solvent removed in vacuo to give2,2,5,5-tetramethyl-[1,3]dioxane-4,6-dione as white solid.

Yield: 6.59 g (79%).

R_(F) (SiO₂, chloroform/ethyl acetate, 98:2): 0.60.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 1.76 (s, 6H); 1.65 (s, 6H).

A solution of 2-(1-Trityl-1H-imidazol-4-yl)-ethyl amine (5.00 g, 14.2mmol) prepared as described above and triethylamine (9.86 mL, 70.7 mmol)in toluene (80 mL) was added dropwise over 50 min to a solution of theabove dione compound (3.65 g, 21.2 mmol) in toluene (40 mL) at 75° C.The mixture was stirred at this temperature for additional 3 hrs (untilthe starting amine was detected on TLC), then it was evaporated todryness. The residue was redissolved in chloroform (300 mL) and washedwith 10% aqueous solution of citric acid (200 mL). The aqueous phase wasextracted with chloroform (2×60 mL); the chloroform phases werecombined, dried over anhydrous magnesium sulfate and solvent removed invacuo. The residue was triturated with hot chloroform (140 mL); hexanes(70 mL) were added and the suspension was stirred at room temperatureovernight. Solids were filtered off, washed with chloroform/hexanesmixture (1:1, 2×50 mL) and dried in vacuo to give the title product.

Yield: 6.73 g (88%).

M.p.: 161-162° C.

R_(F) (SiO₂, chloroform/methanol, 85:15): 0.40.

¹H NMR spectrum (300 MHz, DMSO-d₆, δ_(H)): 12.45 (bs, 1H); 7.66 (t,J=5.1Hz, 1H); 7.57-7.31 (m, 9H); 7.26 (s, 1H); 7.20-7.02 (m, 6H); 6.66(s, 1H); 3.25 (m, 2H); 2.57 (t, J=7.3 Hz, 2H); 1.21 (s, 6H).

4. Synthesis of 4-(4-tert-Butyl-phenyl)-butyric acid

Aluminum chloride powder (80.0 g, 600 mmol) was added in portions to astirred mixture of tert-butylbenzene (40.0 g, 300 mmol) and succinicanhydride (26.7 g, 267 mmol) and 1,1,2,2-tetrachloroethane (100 mL).After all the aluminum chloride had been added, the mixture was pouredinto a mixture of ice (500 mL) and concentrated hydrochloric acid (100mL). The organic layer was separated, washed with water (500 mL) and thesolvent distilled off. Solid residue was dissolved in hot 15% aqueoussolution of sodium carbonate (1000 mL), filtered, cooled and the acidwas precipitated with hydrochloric acid (acidified to pH=1). The crudeacid was filtered, dried on air and recrystalised from benzene (500 mL)to give 4-(4-tert-butyl-phenyl)-4-oxo-butyric acid as colorlesscrystals.

Yield: 36.00 g (58%).

M.p.: 117-120° C.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.93 (dm, J=8.3 Hz, 2H); 7.48(dm, J=8.3 Hz, 2H); 3.30 (t, J=6.6 Hz, 2H); 2.81 (t, J=6.6 Hz, 2H); 1.34(s, 9H).

A mixture of the above acid (36.0 g, 154 mmol), potassium hydroxide(25.8 g, 462 mmol), hydrazine hydrate (20 mL, 400 mmol) and ethyleneglycol (135 mL) was refluxed for 3 hrs, and then distilled until thetemperature of the vapor had risen to 196-198° C. After a further 14 hrsreflux, the mixture was allowed to cool slightly, and was then pouredinto cold water (200 mL). The mixture was acidified with concentratedhydrochloric acid (to pH=1) and extracted with dichloromethane (2×400mL). The organic extracts were combined, dried over anhydrous magnesiumsulfate, solvent removed in vacuo and the residue was purified by columnchromatography (Silicagel 60A, 0.060-0.200 mm; eluent: hexanes/ethylacetate 10:1-6:1) to give the title product as off white solid.

Yield: 16.25 g (48%).

M.p.: 59-60° C.

R_(F) (SiO₂, ethyl acetate): 0.60.

¹H NMR spectrum (300 MHz, CDCl₃, δ0: 7.31 (dm, J=8.1Hz, 2H); 7.12 (dm,J=8.1Hz, 2H); 2.64 (t, J=7.6 Hz, 2H); 2.38 (t, J=7.4 Hz, 2H); 1.96 (m,2H); 1.31 (s, 9H).

5. Synthesis of2,2-Dimethyl-N-(1-trityl-1H-imidazol-4-ylmethyl)-malonamic acid

Hydroxylamine hydrochloride (15.9 g, 229 mmol) was added to a solutionof 4(5)-imidazolecarboxaldehyde (20.0 g, 209 mmol) and sodium carbonate(12.1 g, 114 mmol) in water (400 mL) and the resulting solution wasstirred at room temperature overnight. The mixture was evaporated to 100mL and cooled in an ice bath. The solids were separated by filtrationand the filtrate was concentrated to 40 mL. After cooling to 0° C.,another portion of crystals was obtained. The solids (23 g) werecombined and recrystallised from ethanol (approx. 160 mL) to affordimidazole-4(5)-carbaldehyde oxime as colorless crystals.

Yield: 15.98 g (69%).

¹H NMR spectrum (300 MHz, acetone-d₃+D₂O, δ_(H)): 7.78 (bs, 1H); 7.74(d, J=0.9 Hz, 1H); 7.43 (s, 1H).

Acetyl chloride (51.0 mL, 718 mmol) was added dropwise to methanol (670mL) at 0° C. under argon. After 30 min, the cooling bath was removed andthe above oxime (16.0 g, 144 mmol) was added, followed by palladium oncarbon (5 wt %, 6.1 g). The mixture was hydrogenated at atmosphericpressure for 17 hrs, then it was filtered through Celite and the solventevaporated to give pure 4-(aminomethyl)-imidazole dihydrochloride ascolorless crystals.

Yield: 23.92 g (98%).

¹H NMR spectrum (300 MHz, D₂O, δ_(H)): 8.72 (s, 1H); 7.60 (s, 1H); 4.33(s, 2H). The above amine dihydrochloride (18.9 g; 111 mmol) andtriethylamine (93 mL; 667 mmol) in methanol (1000 mL) were stirred atroom temperature for 10 min. Trifluoroacetic acid ethyl ester (13.3 mL;111 mmol) in methanol (30 mL) was added dropwise over 40 min at 0° C.Reaction mixture was stirred for 18 hrs at room temperature and then itwas evaporated to dryness in vacuo. The residue was dissolved in drydichlormethane (2000 mL) and triethylamine (31 mL; 222 mmol) was added.Then trityl chloride (31.6 g; 113 mmol) was added and the mixture wasstirred overnight at room temperature. Chloroform (1000 mL) and water(1000 mL) were poured into the reaction mixture. Aqueous layer wasseparated and extracted with chloroform (2×300 mL). The combined organiclayers were dried over anhydrous magnesium sulfate. Solvent was removedand the beige solid was triturated with hexanes (1000 mL). Suspensionwas filtered to yield2,2,2-trifluoro-N-(1-trityl-1H-imidazol-4-ylmethyl)-acetamide as whitesolid.

Yield: 46.59 g (96%).

R_(F) (SiO₂, dichloromethane/methanol 95:5): 0.35.

¹H NMR spectrum (300 MHz, DMSO-d₆, δ0: 9.77 (t, J=5.7 Hz, 1H); 7.47-7.34(m, 9H); 7.33 (d, J=1.5 Hz, 1H); 7.13-7.03 (m, 6H); 6.80 (d, J=0.8 Hz,1H); 4.25 (d, J=5.7 Hz, 2H).

The above amide (46.6 g; 107 mmol) was dissolved in tetrahydrofuran (600mL) and ethanol (310 mL). A solution of sodium hydroxide (21.4 g; 535mmol) in water (85 mL) was added. Mixture was stirred for 5 hrs at roomtemperature and then it was concentrated in vacuo. The residue wasseparated between chloroform (1600 mL) and water (800 mL). Aqueous layerwas extracted with chloroform (4×200 mL). Organic layers were combinedand dried over anhydrous magnesium sulfate. Evaporation of the solventyielded (1-trityl-1H-imidazol-4-yl)-methylamine as off white solid.

Yield: 36.30 g (100%).

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.38 (d, J=1.3, 1H); 7.36-7.30(m, 9H); 7.18-7.10 (m, 6H); 6.69 (m, 1H); 3.77 (s, 2H); 1.80 (bs, 2H).

A solution of the above amine (10.0 g, 29.5 mmol) and triethylamine(20.5 mL, 147 mmol) in toluene (220 mL) was added dropwise over 45 minto a solution of 2,2,5,5-tetramethyl-[1,3]dioxane-4,6-dione (3.65 g,21.2 mmol) in toluene (80 mL) at 75° C. The mixture was stirred at thistemperature for additional 3 hrs (until the starting amine was detectedon TLC), then it was evaporated to dryness. The residue was redissolvedin chloroform (500 mL) and washed with 10% aqueous solution of citricacid (300 mL). The aqueous phase was extracted with chloroform (100 mL);the chloroform phases were combined, washed with water (150 mL) driedover anhydrous magnesium sulfate and solvent removed in vacuo. Theresidue was purified by flash column chromatography (silica gel Fluka60, dichloromethane/methanol 98:2 to 9:1) and crystallised fromchloroform/hexanes mixture to give the title product as beige crystals.

Yield: 9.80 g (73%).

M.p.: 174-175° C.

R_(F) (SiO₂, chloroform/methanol, 85:15): 0.35.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 8.45 (t, J=5.8 Hz, 1H); 7.53(s, 1H); 7.40-7.28 (m, 9H); 7.14-7.01 (m, 6H); 6.84 (s, 1H); 4.39 (d,J=5.8 Hz, 2H); 1.44 (s, 6H).

6. Synthesis of 3-(1-Trityl-1H-imidazol-4-yl)-propyl amine

Ethyl 3-(1-trityl-4-imidazolyl)propionate (93.0 g, 223 mmol) intetrahydrofuran/diethyl ether (1:1, 100 mL) was added dropwise to asuspension of lithium aluminium hydride (17.0 g, 446 mmol) during 1 hr.The mixture was refluxed for 3 hrs, then treated with water (100 mL),20% sodium hydroxide (100 mL) and water (100 mL) under cooling withice/water, filtered and the solid washed with tetrahydrofuran. Theorganic phase was dried over anhydrous potassium carbonate, filtered andevaporated to give 3-(1-trityl-4-imidazolyl)propanol as white solid.

Yield: 68.0 g (82%).

M.p.: 127-129° C.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.40-7.24 (m, 10H); 7.17-7.06(m, 6H); 6.55 (s, 1H); 3.72 (t, J=5.3 Hz, 2H); 2.68 (t, J=6.6 Hz, 2H);1.86 (m, 2H).

Methanesulfonyl chloride (8 mL, 104 mmol) was added dropwise to asolution of the above alcohol (32.0 g, 86.8 mmol) in dichloromethane(400 mL) and triethyl amine (15.5 mL) at 0° C. during 1 hr. The mixturewas stirred without cooling for an additional 1 hr; then it was washedwith 5% sodium bicarbonate and dried over anhydrous magnesium sulfate.Dichloromethane was evaporated at 30° C. in vacuo and the residual oilymesylate was used directly in the next step.

Yield: 31.2 g (80%).

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.37-7.30 (m, 10H); 7.16-7.09(m, 6H); 6.58 (s, 1H); 4.24 (t, J=6.3 Hz, 2H); 2.96 (s, 3H); 2.67 (m,2H); 2.10 (m, 2H).

A mixture of the above mesylate (30.0 g, 67 mmol), potassium phtalimide(18.0 g, 100 mmol), sodium iodide (4.0 g, 26.7 mmol) anddimethylformamide (200 mL) was stirred overnight at ambient temperatureand then treated with water (2 L) and benzene (2 L). The organic phasewas dried over anhydrous magnesium sulfate, filtered and solventevaporated giving a residue, which was recrystallised from benzeneyielding 1-trityl-4-(3-phtalimidopropyl)imidazole as white solid.

Yield: 17.2 g (52%).

M.p.: 211-214° C.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.83 (m, 2H); 7.72 (m, 2H);7.39-7.27 (m, 10H); 7.18-7.07 (m, 6H); 6.60 (d, J=0.9 Hz, 1H); 3.72 (t,J=7.4 Hz, 2H); 2.60 (t, J=7.5 Hz, 2H); 1.99 (m, 2H).

The above imidazole derivative (26.6 g, 53.5 mmol) was dissolved inethanol (300 mL) and tetrahydrofuran (150 mL) at 60° C., hydrazinehydrate (50 g, 1 mol) was added and the solution was refluxed for 6 hrsand then heated at 70° C. overnight. The solid was removed by filtrationand the filtrate was treated with 25% aqueous solution of ammonia (2.5I) and dichloromethane (2.5 L). The organic layer was dried overanhydrous potassium carbonate and evaporated to give a residue, whichwas purified by column chromatography on silica gel (Fluka 60,chloroform saturated with ammonia/methanol) giving the title compound aswhite solid.

Yield: 14.2 g (72%).

M.p.: 112-113° C.

R_(F) (SiO₂, chloroform saturated with ammonia/methanol 9:1): 0.30.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.37-7.28 (m, 10H); 7.18-7.09(m, 6H); 6.53 (d, J=1.3 Hz, 1H); 2.74 (t, J=6.9 Hz, 2H); 2.59 (t, J=7.4Hz, 2H); 1.95 (bs, 2H); 1.78 (m, 2H).

7. Synthesis of2,2-Dimethyl-N-[3-(1-trityl-1H-imidazol-4-yl)-propyl]-malonamic acid

2-Chlorotrityl chloride resin (2.3 g, 3.0 mmol) was swelled in DCM for20 mins and filtered. Dimethylmalonic acid (2 eq; 6.0 mmol; 793 mg) wasdissolved i DCM:DMF 1:1 (10 mL) and added to the resin followed by DIPEA(6 eq; 18.0 mmol; 3.14 mL) and DCM (10 mL). The resin was shakenovernight at RT. The resin was filtered and washed with DCM:MeOH:DIPEA(17:2:1), DCM, NMP og DCM (2×25 mL of each). The resin was swelled inDMF for 20 mins and filtered. HOAt (3 eq; 9.0 mmol; 1.23 g), DIC (3 eq;9.0 mmol; 1.40 mL) and DMF (25 mL) was added and the resin was shakenfor 90 min at RT. The resin was filtrered and3-(1-Trityl-1H-imidazol-4-yl)-propyl amine (1.8 eq; 5.40 mmol; 1.84 g),DIPEA (4 eq; 6.0 mmol; 2.09 mL), and DMF (10 mL) was added. The resinwas shaken for 2 days. The resin was filtered and washed with NMP (5×20mL) and DCM (10×20 mL). 2,2,2-Trifluoroethanol/dichlormethan 1:1 (20 mL)was added to the resin and it was shaked for 2 hrs. The resin was washedwith 2,2,2-Trifluoroethanol/dichlormethan 1:1 (10 mL) and the combinedfiltrates were collected and concentrated in vacuo to yield the titlecompound.

Yield: 600 mg (41%).

LCMS4: m/z=482 (M+1)

UPLC (method 02_B4_(—)4): Rt=8.07 min

1H NMR spectrum (300 MHz, DMSO-d_(s), δ_(H)): 7.36-7.44 (9H, m),7.07-7.12 (6H, m), 6.62 (1H, s), 3.02-3.09 (2H, q), 2.38-2.43 (2H, t),1.61-1.69 (2H, m), 1.26 (6H, s).

8. Synthesis of2,2-Dimethyl-N-[3-(1-trityl-1H-imidazol-4-yl)-propyl]-malonamic acidSynthesis of 2,2-Dimethyl-N-pyridin-2-ylmethylmalonamic acid

Chlorotrityl chloride resin (2.3 g, 3.0 mmol) was swelled in DCM for 20mins and filtered. Dimethylmalonic acid (2 eq; 6.0 mmol; 793 mg) wasdissolved i DCM:NMP 1:1 (10 mL) and added to the resin followed by DIPEA(6 eq; 18.0 mmol; 3.14 mL) and DCM (10 mL). The resin was shakenovernight at RT. The resin was filtered and washed with DCM:MeOH:DIPEA(17:2:1), DCM, NMP og DCM (2×25 mL of each). The resin was swelled inNMP for 20 mins and filtered. HOAt (3 eq; 9.0 mmol; 1.23 g), DIC (3 eq;9.0 mmol; 1.40 mL) and NMP (25 mL) was added and the resin was shakenfor 90 min at RT. The resin was filtrered and 2-(Aminomethyl)pyridine (2eq; 6 mmol; 659 mg), DIPEA (4 eq; 6.0 mmol; 2.09 mL), and NMP (10 mL)was added. The resin was shaken for overnigth. The resin was filteredand washed with NMP (5×20 mL) and DCM (10×20 mL). TFA/TIS/water(95:2.5:2.5; 30 mL) was added to the resin and it was shaked for 1 hr,filtered and concentrated in vacuo to yield the title compound.

Yield: 600 mg (41%).

LCMS4: m/z=223 (M+1)

UPLC (method 08_B4_(—)1): Rt=1.79 min

B. Synthesis of Compounds of the Invention Example 1N^(ε26)-[2-(2-{2-[10-(4-Carboxyphenoxy)decanoylamino]ethoxy}ethoxy)acetyl],N^(ε37)-[2-(2-{2-[10-(4-Carboxyphenoxy)decanoylamino]ethoxy}ethoxy)acetyl]-[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)peptide (SEQ ID NO: 7),

Preparation method: SPPS method B, starting with low-loadFmoc-Lys(Mtt)-Wang resin. Fmoc-Lys(Mtt)-OH was used in position 26, andBoc-His(trt)-OH was used in position 7. The Mtt was removed with HFIP,and 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech) and4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared asdescribed in Example 25, step 2 of WO 2006/082204) were coupled using adouble coupling method on the Liberty Peptide synthesiser

UPLC (method 04_A3_(—)1): 10.51 min

LCMS4: m/z=1085.2 (M+4H)⁴⁺, 1447.3 (M+3H)³⁺

Example 2N^(ε26{)2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7),

Preparation method: SPPS method B, starting with low-loadFmoc-Lys(Mtt)-Wang resin. Fmoc-Lys(Mtt)-OH was used in position 26, andBoc-His(Trt)-OH was used in position 7. The Mtt was removed with HFIP,and 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), Fmoc-Glu-OtBu, and4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared asdescribed in Example 25, step 2 of WO 2006/082204) were coupled using adouble coupling method on the Liberty Peptide synthesiser.

UPLC (method 04_A3_(—)1): 7.19 min

LCMS4: m/z=978.5 (M+5H)⁵⁺, 1222.8 (M+4H)⁴⁺1630.1 (M+3H)³⁺

Example 3N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7),

Preparation method: The peptide was synthesised on Lys(Mtt)-Wang resinwith a loading of 0.35 mmol/g. The synthesis was performed on a Libertysynthesiser under microwave conditions using 5 minute single couplingswith DIC/HOAt at up to 70° C., except for histidine which was coupledfor 20 minutes at up to 50° C. All amino acids were protected withstandard protecting groups, except for lysines to be acylated (in thiscase Lys26) which was protected with Mtt. Deprotection was with 5%piperidine in NMP at 50° C. for 3 minutes. After the synthesis wascompleted, the N-terminus was blocked with 10 equivalents ofBoc-carbonate and 10 equivalents of DIPEA for 30 minutes. The Mtt groupswere removed by treatment with neat (undiluted) hexafluoroisopropanolfor 20 minutes and the side chains were built stepwise on the Libertyusing the same protocol as above using Fmoc-8-amino-3,6-dioxaoctanoicacid, Fmoc-Glu-OBut, and hexadecanedioic acid mono-t-butyl ester. Thepeptide was cleaved with TFA/water/TIS (95:2.5:2.5) for 2 hours andisolated by precipitation with diethylether. The crude peptide waspurified by preparative HPLC on a 20 mm×250 mm column packed with either5u or 7u C18 silica. The peptide was dissolved in 5 ml 50% acetic acidand diluted to 20 ml with H₂O and injected on the column which then waseluted with a gradient of 40-60% CH₃CN in 0.1% TFA 10 ml/min during 50min at 40° C. The peptide containing fractions were collected and purityassessed by MALDI and UPLC. The purified peptide was lyophilised afterdilution of the eluate with water.The theoretical molecular mass of 4844.6 was confirmed by MALDI-MS.

UPLC (method 08_B4_(—)1): Rt 9.50 min

UPLC (method 04_A3_(—)1): Rt 11.23 min

Example 4N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7),

Preparation method: As in Example 3, except for the use oftetradecanedioic acid mono-t-butyl ester in the side chain.The theoretical molecular mass of 4788.5 was confirmed by MALDI-MS

UPLC (method 08_B4_(—)1): Rt 8.74 min

UPLC (method 04_A3_(—)1): Rt 9.39 min

Example 5N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],N^(ε37)-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7),

Preparation method: As in Example 3, except for the use of dodecanedioicacid mono-t-butyl ester in the side chain.The theoretical molecular mass of 4732.4 was confirmed by MALDI-MS.

UPLC (method 08_B4_(—)1): Rt 8.19 min

UPLC (method 04_A3_(—)1): Rt 8.17 min

Example 6N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptideamide (SEQ ID NO: 7),

Preparation method: As in Example 3, except that the resin used wasTentagel S RAM with a loading of 0.24 mmol/g and the Fmoc-Lys(Mtt) wasused both on positions 26 and 37.The theoretical molecular mass of 4843.6 was confirmed by MALDI-MS.

UPLC (method 08_B4_(—)1): Rt 9.43 min

UPLC (method 04_A3_(—)1): Rt 11.88 min

Example 7N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptideamide (SEQ ID NO: 7),

Preparation method: As in Example 6, except for the use oftetradecanedioic acid mono-t-butyl ester in the side chain.The theoretical molecular mass of 4787.5 was confirmed by MALDI-MS.

UPLC (method 08_B4_(—)1): Rt 8.72 min

UPLC (method 04_A3_(—)1): Rt 9.98 min

Example 8N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptideamide (SEQ ID NO: 7),

Preparation method: As in Example 6, except for the use of dodecanedioicacid mono-t-butyl ester in the side chain.The theoretical molecular mass of 4731.4 was confirmed by MALDI-MS.

UPLC (method 08_B4_(—)1): Rt 8.16 min

UPLC (method 04_A3_(—)1): Rt 8.83 min

Example 9

N^(ε26)-[2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]ethoxy}ethoxy)acetyl],N^(ε37)-[2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]ethoxy}ethoxy)acetyl][Aib⁸,Arg³⁴,Lys³¹GLP-1(7-37)-peptideamide (SEQ ID NO: 7),

Preparation method: As in Example 7.The theoretical molecular mass of 4497.2 was confirmed by MALDI-MS.

UPLC (method 08_B4_(—)1): Rt 8.85 min

UPLC (method 04_A3_(—)1): Rt 10.27 min

Example 10N^(ε26)-[2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]],N^(ε37)-[2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamino)butyrylamino]][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptideamide (SEQ ID NO: 7),

Preparation method: As in Example 7.The theoretical molecular mass of 4206.8 was confirmed by MALDI-MS.

UPLC (method 08_B4_(—)1): Rt 9.04 min

UPLC (method 04_A3_(—)1): Rt 10.68 min

Example 11N^(ε26)-(2-{2-[2-(2-{2-[2-(13-Carboxy-tridecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl),N^(ε37)-(2-{2-[2-(2-{2-[2-(13-Carboxy-tridecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl)[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide amide (SEQ ID NO: 7),

Preparation method: As in Example 7.The theoretical molecular mass of 4529.2 was confirmed by MALDI-MS.

UPLC (method 08_B4_(—)1): Rt 9.07 min

UPLC (method 04_A3_(—)1): Rt 13.31 min

Example 12N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[4-(4-iodo-phenyl)-butyrylamino]-butyrylamino}-ethoxy)-ethoxy]acetylamino}-ethoxy)-ethoxyFacetyll,N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[4-(4-iodo-phenyl)-butyrylamino]-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7)

Preparation method: SPPS method B,8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), 4-(4-iodophenyl)butyric acid(commercially available from Aldrich) and Fmoc-Glu-OtBu were coupledusing SPPS method D.

UPLC (method 04_A4_(—)1): Rt=8.54 min

UPLC (method 01_A4_(—)2): Rt=10.23 min

LCMS4: Rt=2.4 min, m/z=971(m/5) 1213 (m/44) 1617 (m/3)

Example 13N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[16-(4-carboxyphenoxy)hexadecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[16-(4-carboxyphenoxy)hexadecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}-[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7)

Preparation method: SPPS Method B. The final product was characterisedby analytical UPLC and LC-MS with the exception that an acetic anhydridecapping step was performed after the coupling of the following aminoacids: Trp31, Ala25, Tyr19, Phe12 and Aib8 (2½ min, 65° C. with 1 NAcetic acid anhydride in NMP). The 4-(15-carboxy-pentadecyloxy)benzoicacid tert-butyl ester can be prepared as described in Example 17 inWO07128817.

UPLC (method 08_B4_(—)1): Rt=11.272 min

UPLC (method 05_B10_(—)1): Rt=7.319 min

LCMS4: Rt=2.37 min, m/z=5054.48 Calculated MW=5056.82

Example 14N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[16-(3-carboxyphenoxy)hexadecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[16-(3-carboxyphenoxy)hexadecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7)

Preparation of 3-Hydroxy-benzoic acid tert-butyl ester

A mixture of 3-hydroxybenzoic acid (55.0 g, 400 mmol), di-tert-butyldicarbonate (178 g, 820 mmol), magnesium perchlorate (0.89 g, 4.0 mmol)and dry nitromethane (750 mL) was stirred at 40° C. for 96 hrs. Ethylacetate (800 mL) was added and the organic layer was washed with 5%aqueous solution of sodium bicarbonate (1500 mL). The organic solutionwas dried over anhydrous magnesium sulfate and then evaporated in vacuo.The residue was submitted to column chromatography (silica gel Fluke 60,hexanes/ethyl acetate 8:1) affording the title compound as white solid.

Yield: 9.07 g (12%)

M.p.: 94-96° C.

1H NMR spectrum (300 MHz, CDCl3, ♦_(H) (dH)): 7.61-7.53 (m, 2H); 7.29(t, J=8.1Hz, 1H); 7.05 (m, 1H); 6.06 (bs, 1H); 1.59 (s, 9H).

Preparation of 16-Bromo-hexadecanoic acid methyl ester

16-Bromo-hexadecanoic acid (6.0 g) was dissolved in MeOH (35 mL),toluene (100 mL) and trimethylorthoformate (20 mL), then Amberlyst 15from Fluka (1.4 g) was added. The mixture was stirred at 55° C. for 16h. The mixture was evaporated to dryness and dried under vacuum for 16 hto yield 7.7 g. The residue was suspended in MeOH (ca. 50 mL) andstirred for ca ½ h. The amberlyst 15 was filtered off after stirringwith DCM (30 mL) for ½ h. The filtrate was concentrated to remove theDCM, and the clear solution was cooled and more MeOH (ca 20 mL, total ca40 mL) was added. The flask was cooled and more crystals precipitatedand after stirring for 30 min, the crystals were filtered off and washedwith cold MeOH. The white crystals were dried under vacuum to yield 5.61g.

Preparation of 3-(15-Methoxycarbonyl-pentadecyloxy)-benzoic acidtert-butyl ester

3-Hydroxy-benzoic acid tert-butyl ester (1.79 g) was dissolved in MeCN75 ml, then bromo-hexadecanoic acid methyl ester (3.22 g) was addedfollowed by K₂CO₃ (2.5 g). The reaction was stirred for 3 d at 80° C.The reaction mixture was filtered. The filtrate was evaporated, and theresidue was dissolved in EtOAc 100 ml, and the EtOAc layer was washedtwice with 100 ml brine. The organic layer was dried over MgSO₄ filteredand the solvent was removed by evaporation to give 4.165 g (98%).

Preparation of 3-(15-Carboxy-pentadecyloxy)-benzoic acid tert-butylester

3-(15-Methoxycarbonyl-pentadecyloxy)-benzoic acid tert-butyl ester(4.165 g) was dissolved in 50 ml THF and 50 ml MeOH. Water (10 mL) wasadded followed by LiOH (0.565 g, 13.5 mmol). The reaction was for 16 hat room temperature. The reaction mixture was evaporated and the residuewas dissolved in EtOAC 150 ml, and water 80 and 20 ml of 1 N HCl wasadded. The layers were separated and the organic layer was dried overMgSO₄, filtered and the solvent was removed by evaporation to give awhite solid compound (3.91 g, 97%).

Preparation method: SPPS Method B and using3-(15-Carboxy-pentadecyloxy)-benzoic acid tert-butyl ester in similarfashion as in Example 1. The final product was characterised byanalytical UPLC and LC-MS with the exception that an acetic anhydridecapping step was performed after the coupling of the following aminoacids: Trp31, Ala25, Tyr19, Phe12 and Aib8 (2½ min, 65° C. with 1 NAcetic acid anhydride in NMP).

UPLC (method 08_B4_(—)1): Rt=11.201 min

UPLC (method 05_B10_(—)1): Rt=8.622 min

LCMS4: Rt=2.37 min, m/z=1011.88 (m/5); 1664.32 (m/4); 5053.28

Calculated MW=5056.82

Example 15N^(ε26)-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)-ethoxy]acetyl},N^(ε37)-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]-butyrylamino}ethoxy)ethoxy]acetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7)

Preparation method: SPPS method B, starting with low-loadFmoc-Lys(Mtt)-Wang resin. Fmoc-Lys(Mtt)-OH was used in position 26, andBoc-His(Trt)-OH was used in position 7. The Mtt was removed with HFIP,and 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), Fmoc-Glu-OtBu and4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared asdescribed in Example 25, step 2 of WO 2006/082204) were coupled usingSPPS method D.

UPLC (method 08_B4_(—)1): Rt=8.8 min

UPLC (method 04_A3_(—)1): Rt=9.6 min

LCMS4: 4598.0

Calculated MW=4598.2

Example 16N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[12-(3-carboxyphenoxy)dodecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[12-(3-carboxyphenoxy)dodecanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl][Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7)

Preparation method: SPPS Method B. The 3-(11-carboxy-undecyloxy)-benzoicacid tert-butyl ester was prepared in similar fashion as described for3-(15-carboxy-pentadecyloxy)-benzoic acid tert-butyl ester, empoying12-bromo-dodecanoic acid. The final product was characterised byanalytical UPLC and LC-MS with the exception that an acetic anhydridecapping step was performed after the coupling of the following aminoacids: Trp31, Ala25, Tyr19, Phe12 and Aib8 (2% min, 65° C. with 1 NAcetic acid anhydride in NMP)

UPLC (method 08_B4_(—)1): Rt=9.449 min

LCMS4: Rt=2.37 min, m/z=m/z: 1011.88 (m/4); 1264.32 (m/3); 4942.24

Calculated MW=4944.608

Example 17N^(ε26)-[2-(2-[2-(2-[2-(2-[4-(10-(4-Carboxyphenoxy)decanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]-N^(ε37)-[2-(2-[2-(2-[2-(2-[4-(10-(4-Carboxyphenoxy)decanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Aib⁸,His³¹,Gln³⁴,Lys³⁷]GLP-1(7-37)-peptide (SEQ IDNO: 3)

Preparation: SPPS method A, starting with low-load Fmoc-Lys(Mtt)-Wangresin. Fmoc-Lys(Mtt)-OH was used in position 26, and Boc-His(trt)-OH wasused in position 7. The Mtt was removed with HFIP, and8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech) was coupled twice followed byFmoc-Glu-OtBu and 4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester(prepared as described in Example 25, step 2 of WO 2006/082204) werecoupled using SPPS method A.

UPLC (method 05_B5_(—)1): Rt=4.95 min (92%)

LCMS4: m/z=4011, calculated=4011

Example 18N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[4-(4-methylphenyl)butyrylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[4-(4-methylphenyl)butyrylamino]butyrylamino}ethoxy)ethoxy]-acetylamino}ethoxy)ethoxy]acetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7)

Preparation: SPPS method B,8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), 4-(4-methylphenyl)butyricacid (commercially available from ABCR) and Fmoc-Glu-OtBu were coupledusing SPPS method D.

UPLC (method 01_B4_(—)1): Rt=9.93 min

LCMS4: Rt=2.44 min, m/z=926(m/5) 1157(m/4) 1543(m/3)

Example 19N^(ε26)-((S)-4-Carboxy-4-{(S)-4-carboxy-4-[10-(4-carboxyphenoxy)-decanoylamino]butyrylamino}butyryl),N^(ε37)-((S)-4-Carboxy-4-{(S)-4-carboxy-4-[10-(4-carboxyphenoxy)-decanoylamino]butyrylamino}butyryl)[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7)

Preparation: SPPS method B, starting with low-load Fmoc-Lys(Mtt)-Wangresin. Fmoc-Lys(Mtt)-OH was used in position 26, and Boc-His(Trt)-OH wasused in position 7. The Mtt was removed with HFIP, and Fmoc-Glu-OtBu and4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared asdescribed in Example 25, step 2 of WO 2006/082204) were coupled usingSPPS method D.

UPLC (method 08_B4_(—)1): Rt=8.6 min

UPLC (method 04_A3_(—)1): Rt=7.9 min

LCMS4: 4565.0

Calculated MW=4566.1

Example 20N^(ε26)-{2-[2-(2-{2-[2-(2-{4-Carboxy-4-[10-(3-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},N^(ε37)-{2-[2-(2-{2-[2-(2-{4-Carboxy-4-[10-(3-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7)

Preparation of 3-(9-Carboxy-nonyloxy)benzoic acid tert-butyl ester

3-Hydroxy-benzoic acid tert-butyl ester (3 g) was dissolved inacetonitrile (50 mL). 10-Bromo decanoic acid methyl ester from Aldrich(4.1 g) in acetonitrile (20 mL) was added and washing the vessel withacetonitrile (30 mL). Potassium carbonate was added and the mixture wasrefluxed under nitrogen for ca. 18 h. The reaction was cooled andevaporated to dryness. The residue was dissolved in AcOEt (80 mL) andwater (30 mL) and extracted. The aqueous phase was washed with AcOEt (30mL) and the combined organic phases were washed with water (50 mL), sat.NaCl (30 mL) and dried over MgSO₄ and the filtrate was concentratedunder vacuum to yield a white solid (5.8 g). The residue was dissolvedin DCM (15 mL) and heptane (ca 60 mL) was added, and the solution wasconcentrated to ca. 30 mL. After stirring for 30 min. crystals began toform, and the solution was ice-cooled. The crystals were filtered offand washed with cooled heptane and dried in under vacuum to yield 4.13 g(71%) of 3-(9-methoxycarbonyl-nonyloxy)-benzoic acid tert-butyl ester.

The crystals were dissolved in THF (30 mL) and 1N NaOH (11 mL) wasadded. The turbid solution was stirred for 16 h. The reaction mixturewas concentrated to remove the majority of THF, and remaining aqueoussolution was extracted with AcOEt (50 mL). The pH of the aqueoussolution was adjusted to 1-2 with ca. 12 mL 1 N HCl, and the aqueousphase was extracted with AcOEt (25 mL). The combined organic phases werewashed with water, dried over MgSO₄, filtered and concentrated to yielda white semi-crystalline solid (3.97 g). LCMS2: 401 (M+23), H-NMR (400MHz, CDCl₃): 7.56 (d, 1H), 7.50 (m, 1H), 7.26-7.32 (m, 1H), 7.05 (dd,1H), 3.99 (t, 2H), 2.35 (t, 2H), 1.75-1.82 (m, 2H), 1.62-1.65 (m, 2H),1.59 (s, 9H), 1.42-1.47 (m, 2H), 1.33 (br, 8H).

Preparation method: SPPS Method B, starting with low-loadFmoc-Lys(Mtt)-Wang resin. Fmoc-Lys(Mtt)-OH was used in position 26, andBoc-His(trt)-OH was used in position 7. The

Mtt was removed with HFIP, and8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), Fmoc-Glu-OtBu, and3-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester were coupled usinga double coupling method on the Liberty Peptide synthesiser.

UPLC (method 04_A4_(—)1): 10.01 min

UPLC (method 08_B4_(—)1): 8.81 min

LCMS4: m/z=978.5 (M+5H)⁵⁺, 1222.8 (M+4H)⁴⁺, 1630.1 (M+3H)³⁺

Example 21N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy]ethoxy}acetyl][Aib⁸,His³¹,Gln³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 3)

Preparation method: As in Example 5.The theoretical molecular mass of 4655.2 was confirmed by MALDI

UPLC (method 08_B4_(—)1): Rt=7.72 min

UPLC (method 04_A3_(—)1): Rt=5.70 min

Example 22N⁹-{2-[2-(1H-Imidazol-4-yl)ethylcarbamoyl]-2-methylpropionyl},N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}[Arg³⁴, Lys³⁷GLP-1(9-37)Glu³⁸-peptide (SEQ ID NO: 2)

Preparation: SPPS method B.2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid wascoupled using the same coupling condition as an Aib amino acis.8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), Fmoc-Glu-OtBu , and4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared asdescribed in Example 25, step 2 of WO 2006/082204) were coupled using

SPPS method D.

UPLC (method 04_A3_(—)1): Rt=9.32 min.

LCMS4: Rt=2.29 min., m/z=1669 (m/3), 1252 (m/4), 1001 (m/5)

Example 23N⁹-{2-[2-(1H-Imidazol-4-yl)ethylcarbamoyl]-2-methylpropionyl}-N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}[Arg³⁴, Lys³⁷]GLP-1(9-37)-peptide (SEQ ID NO: 11)

Preparation: SPPS method B,2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid wascoupled using the same coupling condition as an Aib amino acid.8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), Fmoc-Glu-OtBu and4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared asdescribed in Example 25, step 2 of WO 2006/082204) were coupled usingSPPS method D.

UPLC (method 08_B4_(—)1 (TFA)): Rt=8.81 min

LCMS4: Rt=2.29 min, m/z=1625 (m/3), 1219 (m/4), 975 (m/5)

Example 24N^(ε26)-{2-[2-(2-{(S)-4-Carboxy-4-[2-(2-{2-[(13-carboxytridecanoylamino)]ethoxy}ethoxy)acetylamino]butyrylamino}ethoxy)ethoxy]acetyl},N^(ε37)-{2-[2-(2-{(S)-4-Carboxy-4-[2-(2-{2-[(13-carboxytridecanoylamino)]ethoxy}ethoxy)acetylamino]butyrylamino}ethoxy)ethoxy]acetyl}-[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7)

Preparation: SPPS method B, starting with low-load Fmoc-Lys(Mtt)-Wangresin. Fmoc-Lys(Mtt)-OH was used in position 26, and Boc-His(trt)-OH wasused in position 7. The Mtt was removed with HFIP manually, and8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), Fmoc-Glu-OtBu andtetradecanedioc were coupled using a double coupling method on theLiberty Peptide synthesiser. The theoretical molecular mass wasconfirmed by MALDI-MS.

UPLC (method 08_B4_(—)1): Rt=8.6 min

UPLC (method 04_A3_(—)1): Rt=9.7 min

MALDI-MS: 4788

Example 25N^(ε26)-[(S)-4-Carboxy-4-{2-[2-(2-[2-(2-{2-[(13-carboxytridecanoylamino)]ethoxy}ethoxy)acetylamino]ethoxy)ethoxy]acetylamino}butyryl],N^(ε37)-[(S)-4-Carboxy-4-{2-[2-(2-[2-(2-{2-[(13-carboxytridecanoylamino)]ethoxy}ethoxy)acetylamino]ethoxy)ethoxy]acetylamino}butyryl][Aib⁸,Arq³⁴,Lys³⁷]GLP-1(7-37)-peptide(SEQ ID NO: 7)

Preparation: SPPS method B, starting with low-load Fmoc-Lys(Mtt)-Wangresin. Fmoc-Lys(Mtt)-OH was used in position 26, and Boc-His(trt)-OH wasused in position 7. The Mtt was removed with HFIP manually, and8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), Fmoc-Glu-OtBu andtetradecanedioc were coupled using a double coupling method on theLiberty Peptide synthesiser. The theoretical molecular mass wasconfirmed by MALDI-MS.

UPLC (method 08_B4_(—)1): Rt=8.8 min

UPLC (method 04_A3_(—)1): Rt=10 min

MALDI-MS: 4787

Example 26N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-[4-(4-tert-Butyl-phenyl)-butyrylamino]-4-carboxy-butyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-[4-(4-tert-Butyl-phenyl)-butyrylamino]-4-carboxy-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl}[Aib⁸,Arg³⁴,Lys³⁷]GLP-1(7-37)-peptide (SEQ ID NO: [[9]]7), Chem. 45

Preparation: SPPS method B,8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), 4-(4-t-butylphenyl)butyricacid and Fmoc-Glu-OtBu were coupled using SPPS method D.

UPLC (method 08_B4_(—)1): Rt=9.07 min

LCMS4: Rt=2.29 min, m/z=943 (m/5) 1179 (m/4) 1571 (m/3)

Example 27N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methylpropionyl}-N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-[4-(4-tert-Butylphenyl)butyrylamino]-4-carboxybutyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl},N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-[4-(4-tert-Butylphenyl)butyrylamino]-4-carboxybutyrylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl][Arg³⁴,Lys³¹]GLP-1(9-37)-peptide (SEQ ID NO: 11)

Preparation: SPPS method B,2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid wascoupled using the same coupling condition as Fmoc-Aib amino acid.8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), Fmoc-Glu-OtBu and4-(4-t-butylphenyl)butyric acid were coupled using SPPS method D.

UPLC (method 04_A4_(—)1): Rt=10.56 min

LCMS4: Rt=2.40 min. m/z=940(m/5), 1174(m/4), 1565(m/3)

Example 28N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Imp⁷,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 12)

Preparation method: SPPS method B

LCMS4: Rt: 2.22 min, m/z: 4859.5; 1214.9 (M+4H)⁴⁺; 1619.8 (M+3H)³⁺

UPLC (method: 08_B4_(—)1): Rt=8.88 min

UPLC (method: 04_A3_(—)1): Rt=9.28 min

Example 29N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 9)

Preparation method: SPPS method B

UPLC (method 05_B5_(—)1): Rt=5.75 min

UPLC (method 08_B2_(—)1): Rt=13.09 min

LCMS4 (M/5)+1=976; (M/4)+1=1219; Exact mass=4874

Example 30N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl][Aib8,Gly9,Arg34,Lys37]-GLP-1-(7-37)-peptide(SEQ ID NO: 5)

Preparation method: SPPS method A

UPLC (method 09_B2_(—)1): Rt=13.20 min

UPLC (method 05_B5_(—)1): Rt=6.05 min

LCMS4: (M/5)+1=964; (M/4)+1=1204; Exact mass=4816

Example 31N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl][Aib⁸,Arg²³,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 4)

Preparation method: SPPS method B

LCMS4: Rt=2.12 min, m/z: 4916.0

UPLC (method: 08_B2_(—)1): Rt=12.59 min

UPLC (method: 04_A3_(—)1): Rt=10.57 min

Example 32N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amno]ethoxy]ethoxy]acetyl]-[Arg34,Lys37]-GLP-1-(7-37)-peptide (SEQ ID NO:9)

Preparation method: SPPS method B

LCMS4: Rt=2.12 min, m/z: 4774.4

UPLC (method: 09_B2_(—)1): Rt=12.87 min

UPLC (method: 04_A3_(—)1): Rt=8.86 min

Example 33N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl][His³¹,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 10)

Preparation method: SPPS method B

LCMS4: Rt: =1.92 min, m/z: 4797.3; M/4: 1199.8; M/3: 1599.4

UPLC (method: 09_B4_(—)1): Rt=8.12 min

UPLC (method: 05_B8_(—)1): Rt=2.03 min

Example 34N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[His³¹,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 10)

Preparation method: SPPS method B

LCMS4: Rt=1.99 min, m/z: 4697.0

UPLC (method: 09_B2_(—)1) Rt=12.20 min

UPLC (method: 05_B5_(—)1): Rt=5.31 min

Example 35N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(11-carboxyundecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(11-carboxyundecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[His³¹,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 10)

Preparation method: SPPS method B

LCMS4: Rt=1.89 min, m/z: 4641.2

UPLC (method: 09_B2_(—)1): Rt=11.2 min

UPLC (method: 05_B5_(—)1): Rt=4.00 min

Example 36N^(ε26)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[10-(4-carboxyphenoxy)decanoylamino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl],N^(ε37)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[10-(4-carboxyphenoxy)decanoylamino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl][His³¹,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 10)

Preparation method: SPPS method B

LCMS4: Rt: 1.97 min, m/z: 4797.3; M/4: 1199.8; M/3: 1599.4

UPLC (method: 09_B4_(—)1): Rt=8.24 min

UPLC (method: 05_B8_(—)1): Rt=2.88 min

Example 37N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl][Gln⁹,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 8)

Preparation method: SPPS method B

LCMS4: Rt=1.06 min, m/z: 4873.3

UPLC (method: 09_B2_(—)1): Rt=13.18 min

UPLC (method: 05_B5_(—)1): Rt=6.40 min

Example 38

N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetylF[Glu³⁰,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide (SEQ ID NO: 13),

Preparation method: SPPS method B

LCMS4: Rt=2.13 min, m/z: 4932.7

UPLC (method: 09_B2_(—)1): Rt=13.39 min

UPLC (method: 04_A3_(—)1): Rt=8.20 min

Example 39N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[8-(4-carboxyphenoxy)octanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[8-(4-carboxyphenoxy)octanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 7)

Preparation method: SPPS method B

LCMS4: Rt: 1.93 min, m/z: 4832.4; M/4: 1208.5; M/3: 1611.0

UPLC (method 09_B4_(—)1): Rt=8.10 min

UPLC (method 04_A3_(—)1): Rt=8.15 min

UPLC (method 05_B5_(—)1): Rt=5.30 min

Example 40N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[8-(4-carboxyphenoxy)octanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[8-(4-carboxyphenoxy)octanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 9)

Preparation method: SPPS method B

LCMS4: Rt: 1.92 min, m/z: 4818.4; M/4: 1205.0; M/3: 1606.7

UPLC (method 09_B4_(—)1): Rt=8.06 min

UPLC (method 04_A3_(—)1): Rt=8.02 min

Example 41N{9}-[2,2-dimethyl-3-oxo-3-(pyridin-2-ylmethylamino)propanoyl],N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetylF[Arg³⁴,Lys³⁷]-GLP-1-(9-37)-peptide (SEQ ID NO: 11)

Preparation method: SSPS method B.2,2-Dimethyl-N-pyridin-2-ylmethyl-malonamic acid was coupled using thesame coupling condition as used for2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethylFinalonamic acid inthe previous examples. Fmoc-Glu-OtBu and 4-(9-carboxy-nonyloxy)-benzoicacid tert-butyl ester (prepared as described in Example 25, step 2 of WO2006/082204) were coupled using SPPS method D.

UPLC (method 08_B4_(—)1): Rt=8.98 min

LCMS4: Rt=2.23 min. m/z=1624(m/3), 1218 (m/4)

Example 42N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamno] butanoyl]a m ino]ethoxy]ethoxy]acetyl]a mino]ethoxy]ethoxy]acetyl]-[Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptidyl-Gly (SEQ IDNO: 9)

Preparation method: SSPS method B

LCMS4: Rt: 2.05 min, m/z: 4931.5; M/4: 1233.3; M/3: 1644.4

UPLC (method 09_B4_(—)1): Rt=8.52 min

UPLC (method 05_B5_(—)1): Rt=5.18 min

UPLC (method 04_A3_(—)1): Rt=9.24 min

Example 43N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg³⁴,Gly³⁶,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 6)

Preparation method: SSPS method B

LCMS4: Rt: 2.18 min, m/z: 4775.3; M/4: 1194.5; M/3: 1592.4

UPLC (method: 09_B4_(—)1): Rt=9.01 min

UPLC (method: 04_A3_(—)1): Rt=9.60 min

UPLC (method: 05_B5_(—)1): Rt=5.88 min

Example 44N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[9-(4-carboxyphenoxy)nonanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[9-(4-carboxyphenoxy)nonanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 9)

Preparation method: SSPS method B

LCMS4: Rt: 2.03 min, m/z: 4846.4; M/4: 1212.3; M/3: 1616.1

UPLC (method: 09_B4_(—)1): Rt=8.27 min

UPLC (method: 05_B5_(—)1): Rt=5.09 min

Example 45N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[13-(5-carboxythiophene-2-yl)tridecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-[13-(5-carboxythiophene-2-yl)tridecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 7)

Preperation method: SSPS method B.8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), Fmoc-Glu-OtBu, and5-(12-Carboxy-dodecyl)-thiophene-2-carboxylic acid tert-butyl ester(prepared as described in Example 6 of WO07128815) were coupled usingSSPS method D method on the Liberty synthesiser.

UPLC (method 08_B4_(—)1): Rt=9.87 min

LCMS4: m/z=1651(m/3), 1239 (m/4), 991 (m/5)

Example 46N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptidyl-Glu(SEQ ID NO: 14), Chem. 65

Preparation method: SPPS method A

UPLC (method 10_B14_(—)1): Rt=6.54 min

LCMS4: (M/5)+1=1001; (M/4)+1=1251; Exact mass=5003.5

Example 47N^(ε26)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl],N^(ε37)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoylF[Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide (SEQ ID NO: 9)

Preparation method: SSPS method B

UPLC (method: 09_B4_(—)1): Rt=8.76 min.

UPLC (method: 04_A6_(—)1): Rt=6.02 min.

LCMS4: Rt=2.12 min. m/z: 4775; M/4=1194; M/5=955

Example 48N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(2S)-4-carboxy-2-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(2S)-4-carboxy-2-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 7)

Preparation method: SSPS method B

UPLC (method:08_B2_(—)1): Rt=13.193 min

UPLC (method:05_B5_(—)1): Rt=6.685 min

LCMS4: m/z: 4887; m/3:1630; m/4:1222; m/5:978

Example 49N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[8-(4-carboxyphenoxy)octanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[8-(4-carboxyphenoxy)octanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg34,Gly36,Lys37]-GLP-1-(7-37)-peptide(SEQ ID NO: 6)

Preparation method: SSPS method B

LCMS4: Rt: 2.07 min, m/z: 4719.2; M/4: 1180.5; M/3: 1573.7

UPLC (method: 08_B4_(—)1): Rt=8.45 min

UPLC (method: 05_B5_(—)1): Rt=5.19 min

Pharmacological Methods Example 50 In Vitro Potency

The purpose of this example is to test the activity, or potency, of theGLP-1 derivatives in vitro.

The potencies of theef GLP-1 derivatives of Examples 1-49 weredetermined as described below, i.e. as the stimulation of the formationof cyclic AMP (cAMP) in a medium containing membranes expressing thehuman GLP-1 receptor.

Principle

Purified plasma membranes from a stable transfected cell line,BHK467-12A (tk-ts13), expressing the human GLP-1 receptor werestimulated with the GLP-1 analogue or derivative in question, and thepotency of cAMP production was measured using the AlphaScreen™ cAMPAssay Kit from Perkin Elmer Life Sciences. The basic principle of TheAlphaScreen Assay is a competition between endogenous cAMP andexogenously added biotin-cAMP. The capture of cAMP is achieved by usinga specific antibody conjugated to acceptor beads.

Cell Culture and Preparation of Membranes

A stable transfected cell line and a high expressing clone were selectedfor screening. The cells were grown at 5% CO₂ in DMEM, 5% FCS, 1%Pen/Strep (Penicillin/Streptomycin) and 0.5 mg/ml of the selectionmarker G418.

Cells at approximate 80% confluence were washed 2× with PBS andharvested with Versene (aqueous solution of the tetrasodium salt ofethylenediaminetetraacetic acid), centrifuged 5 min at 1000 rpm and thesupernatant removed. The additional steps were all made on ice. The cellpellet was homogenised by the Ultrathurax for 20-30 sec. in 10 ml ofBuffer 1 (20 mM Na-HEPES, 10 mM EDTA, pH=7.4), centrifuged 15 min at20,000 rpm and the pellet resuspended in 10 ml of Buffer 2 (20 mMNa-HEPES, 0.1 mM EDTA, pH=7.4). The suspension was homogenised for 20-30sec and centrifuged 15 min at 20,000 rpm. Suspension in Buffer 2,homogenisation and centrifugation was repeated once and the membraneswere resuspended in Buffer 2. The protein concentration was determinedand the membranes stored at −80° C. until use.

The assay was performed in ½-area 96-well plates, flat bottom (Costarcat. no: 3693). The final volume per well was 50 μl.

Solutions and Reagents

AlphaScreen cAMP Assay Kit from Perkin Elmer Life Sciences (cat. No:6760625M); containing Anti-cAMP Acceptor beads (10 U/μl), StreptavidinDonor beads (10 U/μl) and Biotinylated-cAMP (133 U/μl).

AlphaScreen Buffer, pH=7.4: 50 mM TRIS-HCl (Sigma, cat. no: T3253); 5 mMHEPES (Sigma, cat. no: H3375); 10 mM MgCl₂, 6H₂O (Merck, cat. no: 5833);150 mM NaCl (Sigma, cat. no: S9625); 0.01% Tween™ (Merck, cat. no:822184). The following was added to the AlphaScreen Buffer prior to use(final concentrations indicated): BSA (Sigma, cat. no. A7906): 0.1%;IBMX (Sigma, cat. no. 15879): 0.5 mM; ATP (Sigma, cat. no. A7699): 1 mM;GTP (Sigma, cat. no. G8877): 1 uM.

cAMP standard (dilution factor in assay=5): cAMP Solution: 5 μL of a 5mM cAMP-stock+495 μL AlphaScreen Buffer.

Suitable dilution series in AlphaScreen Buffer were prepared of the cAMPstandard as well as the GLP-1 analogue or derivative to be tested, e.g.the following eight concentrations of the GLP-1 compound: 10⁻⁷, 10⁻⁸,10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ and 10⁻¹⁴M, and a series from, e.g.,10⁻⁶ to 3×10⁻¹¹ of cAMP.

Membrane/Acceptor Beads

Use hGLP-1/BHK 467-12A membranes; 6 μg/well corresponding to 0.6 mg/ml(the amount of membranes used pr. well may vary)

“No membranes”: Acceptor Beads (15 μg/mlfinal) in AlphaScreen buffer

“6 μg/well membranes”: membranes+Acceptor Beads (15 μg/mlfinal) inAlphaScreen buffer

Add 10 μl “No membranes” to the cAMP standard (per well in duplicates)and the positive and negative controls

Add 10 μl “6 μg/well membranes” to GLP-1 and analogues (per well induplicates/triplicates)

Pos. Control: 10 μl “no membranes”+10 μl AlphaScreen Buffer

Neg. Control: 10 μl “no membranes”+10 μl cAMP Stock Solution (50 μM)

As the beads are sensitive to direct light, any handling was in the dark(as dark as possible), or in green light. All dilutions were made onice.

Procedure

1. Make the AlphaScreen Buffer.

2. Dissolve and dilute the GLP-1/Analogues/cAMP standard in AlphaScreenBuffer.

3. Make the Donor Beads solution and incubate 30 min. at RT.

4. Add the cAMP/GLP-1/Analogues to the plate: 10 μl per well.

5. Prepare membrane/Acceptor Beads solution and add this to the plates:10 μl per well.

6. Add the Donor Beads: 30 piper well.

7. Wrap the plate in aluminum foil and incubate on the shaker for 3hours (very slowly) at RT.

8. Count on AlphaScreen—each plate pre incubates in the AlphaScreen for3 minutes before counting.

Results

The EC₅₀ [pM] values were calculated using the Graph-Pad Prism software(version 5).

The potency of all derivatives in vitro was confirmed. 43 derivativeshad a good in vitro potency corresponding to an EC₅₀ of 2000 pM orbelow; 42 derivatives were even more potent having an EC₅₀ at 1000 pM orbelow; 35 derivatives had a still further improved potency correspondingto an EC₅₀ at 500 pM or below; 19 derivatives were very potent,corresponding to an EC₅₀ at 200 pM or below; and 10 derivatives had avery good potency corresponding to an EC₅₀ at 100 pM or below. Forcomparison, compound no. 13 in Table 1 of Journal of Medicinal Chemistry(2000), vol. 43, no. 9, p. 1664-669 (GLP-1(7-37) acylated at K^(26,34)with bis-C12-diacid) had an in vitro potency correspondingto an EC₅₀ of1200 pM.

If desired, the fold variation in relation to GLP-1 may be calculated asEC₅₀ (GLP-1)/EC₅₀ (analogue)−3693.2.

Example 51 GLP-1 Receptor Binding

The purpose of this experiment is to investigate the binding to theGLP-1 receptor of the GLP-1 derivatives, and how the binding ispotentially influenced by the presence of albumin. This is done in an invitro experiment as described below.

The binding affinity of the GLP-1 derivatives of Examples 1-49 to thehuman GLP-1 receptor was measured by way of their ability to displace of¹²⁵I-GLP-1 from the receptor. Liraglutide and semaglutide were includedas comparative compounds. In order to test the binding of thederivatives to albumin, the assay was performed with a low concentrationof albumin (0.005%—corresponding to the residual amount thereof in thetracer), as well as with a high concentration of albumin (2.0% added). Ashift in the binding affinity, IC₅₀, is an indication that the peptidein question binds to albumin, and thereby a prediction of a potentialprotracted pharmacokinetic profile of the peptide in question in animalmodels.

Conditions

Species (in vitro): Hamster

Biological End Point: Receptor Binding

Assay Method: SPA

Receptor: GLP-1 receptor

Cell Line: BHK tk-ts13

Cell Culture and Membrane Purification

A stable transfected cell line and a high expressing clone were selectedfor screening. The cells were grown at 5% CO₂ in DMEM, 10% FCS, 1%Pen/Strep (Penicillin/Streptomycin) and 1.0 mg/ml of the selectionmarker G418.

The cells (approx. 80% confluence) were washed twice in PBS andharvested with Versene (aqueous solution of the tetrasodium salt ofethylenediaminetetraacetic acid), following which they were separated bycentrifugation at 1000 rpm for 5 min. The cells/cell pellet must be kepton ice to the extent possible in the subsequent steps. The cell pelletwas homogenised with Ultrathurrax for 20-30 seconds in a suitable amountof Buffer 1 (depending on the amount of cells, but e.g. 10 ml). Thehomogenate was centrifuged at 20000 rpm for 15 minutes. The pellet wasresuspended (homogenised) in 10 ml Buffer 2 and re-centrifuged. Thisstep was repeated once more. The resulting pellet was resuspended inBuffer 2, and the protein concentration was determined. The membraneswere stored at minus 80° C.

Buffer 1: 20 mM Na-HEPES+10 mM EDTA, pH 7.4

Buffer 2: 20 mM Na-HEPES+0.1 mM EDTA, pH 7.4

Binding Assay:

SPA:

Test compounds, membranes, SPA-particles and [¹²⁵I]]-GLP-1(7-36)NH₂ werediluted in assay buffer. 25 ul (micro liter) of test compounds wereadded to Optiplate. HSA (“high albumin” experiment containing 2% HSA),or buffer (“low albumin” experiment containing 0.005% HSA), was added(50 ul). 5-10 ug protein/sample was added (50 ul) corresponding to0.1-0.2 mg protein/ml (to be preferably optimised for each membranepreparation). SPA-particles (Wheatgerm agglutinin SPA beads, PerkinElmer, #RPNQ0001) were added in an amount of 0.5 mg/well (50 ul). Theincubation was started with [¹²⁵I]-GLP-1]-(7-36)NH₂ (final concentration0.06 nM corresponding to 49.880 DPM, 25 ul). The plates were sealed withPlateSealer and incubated for 120 minutes at 30° C. while shaking. Theplates were centrifuged (1500 rpm, 10 min) and counted in Topcounter.

Assay Buffer:

50 mM HEPES

5 mM EGTA

5 mM MgCl2

0.005% Tween™ 20

pH 7.4

HSA was SIGMA A1653.

Calculations

The IC₅₀ value was read from the curve as the concentration whichdisplaces 50% of ¹²⁵I-GLP-1 from the receptor, and the ratio of[(IC₅₀/nM) high HSA]/[(IC₅₀/nM) ultralow HSA] was determined.

Generally, the binding to the GLP-1 receptor at low albuminconcentration should be as good as possible, corresponding to a low IC₅₀value.

The IC₅₀ value at high albumin concentration is a measure of theinfluence of albumin on the binding of the derivative to the GLP-1receptor. As is known, the GLP-1 derivatives also bind to albumin. Thisis a generally desirable effect, which extends their lifetime in plasma.Therefore, the IC₅₀ value at high albumin will generally be higher thanthe IC₅₀ value at low albumin, corresponding to a reduced binding to theGLP-1 receptor, caused by albumin binding competing with the binding tothe GLP-1 receptor.

A high ratio (IC₅₀ value (high albumin)/IC₅₀ value (low albumin)) maytherefore be taken as an indication that the derivative in questionbinds well to albumin (may have a long half-life), and also per se bindswell to the GLP-1 receptor (the IC₅₀ value (high albumin) is high, andthe IC₅₀ value (low albumin) is low).

Results

The following results were obtained, where “ratio” refers to [(IC₅₀/nM)high HSA]/[(IC₅₀/nM) low HSA]):

All but two derivatives had a ratio above 1.0; 40 derivatives were above10; 34 derivatives were above 25; 22 derivatives were above 50; 12derivatives above 100; and 3 derivatives had a ratio above 250.

Furthermore as regards IC₅₀ (low albumin), all derivatives had an IC₅₀(low albumin) below 600 nM; all but one were below 500 nM; 46derivatives were below 100 nM; 44 derivatives were below 50.00 nM; 34derivatives were below 10.00 nM; 23 derivatives were below 5.00 nM; and7 derivatives were below 1.00 nM.

Finally as regards IC₅₀ (high albumin), all derivatives had an IC₅₀(high albumin) at 1000.00 nM or below; 46 derivatives were below 1000.00nM; 39 derivatives were below 500.00 nM; 7 derivatives were below 100.00nM; and 4 derivatives were below 50.00 nM.

Example 52 Estimate of Oral Bioavailability

The purpose of this experiment is to estimate the oral bioavailabilityof the GLP-1 derivatives.

To this end, the exposure in plasma after direct injection into theintestinal lumen of the GLP-1 derivatives of Examples 2, 15-17, 21, 25,32, 36-39, and 42-48 was studied in vivo in rats, as described in thefollowing.

The GLP-1 derivatives were tested in a concentration of 1000 uM in asolution of 55 mg/ml sodium caprate.

32 male Sprague Dawley rats with a body weight upon arrival ofapproximately 240 g were obtained from Taconic (Denmark) and assigned tothe different treatments by simple randomisation, 4 rats per group. Therats were fasted for approximately 18 hours before the experiment andtaken into general anaesthesia (Hypnorm/Dormicum).

The GLP-1 derivatives were administered in the jejunum either in theproximal part (10 cm distal for the duodenum) or in the mid-intestine(50 cm proximal for the cecum). A PE50-catheter, 10 cm long was insertedinto the jejunum, forwarded at least 1.5 cm into the jejunum, andsecured before dosing by ligature around the gut and the catheter with3/0 suture distal to tip to prevent leak or catheter displacement.Catheter was placed without syringe and needle and 2 ml saline wasadministered into abdomen before closing the incision with wound clips.

100 μl of the respective GLP-1 derivative was injected into the jejunallumen through the catheter with a 1 ml syringe. Subsequently, 200 μl ofair was pushed into the jejunal lumen with another syringe to “flush”the catheter. This syringe was leaved connected to the catheter toprevent flow back into the catheter.

Blood samples (200 ul) were collected at desired intervals (usually attimes 0, 10, 30, 60, 120 and 240 min) into EDTA tubes from the tail veinand centrifuged 5 minutes, 10000 G, at 4° C. within 20 minutes. Plasma(75u1) was separated to Micronic tubes, immediately frozen, and kept at−20° C. until analyzed for plasma concentration of the respective GLP-1derivative with LOCI (Luminescent Oxygen Channeling Immunoassay),generally as described for the determination of insulin by Poulsen andJensen in Journal of Biomolecular Screening 2007, vol. 12, p. 240-247.The donor beads were coated with streptavidin, while acceptor beads wereconjugated with a monoclonal antibody recognising a mid-/C-terminalepitope of the peptide. Another monoclonal antibody, specific for theN-terminus, was biotinylated. The three reactants were combined with theanalyte and formed a two-sited immuno-complex. Illumination of thecomplex released singlet oxygen atoms from the donor beads, which werechanneled into the acceptor beads and triggered chemiluminescence whichwas measured in an Envision plate reader. The amount of light wasproportional to the concentration of the compound.

After the blood sampling the rats were sacrificed under anaesthesia andthe abdomen was opened to verify correct catheter placement.

The mean (n=4) plasma concentrations (pmol/l) were determined as afunction of time. The ratio of plasma concentration (pmol/l) divided bythe concentration of the dosing solution (pmol/l) was calculated foreach treatment, and the results for t=30 min (30 minutes after theinjection of the compound in the jejunum) were assessed (dose-correctedexposure at 30 min) as a surrogate measure of intestinalbioavailability. The dose-corrected exposure has been shown to correlatesignificantly with the actual bioavailability.

The following results were obtained, where dose-corrected exposure at 30min refers to (the plasma concentration 30 minutes after injection ofthe compound in the jejunum (pM)), divided by (the concentration of thecompound in the dosing solution (μM)):

All derivatives had a dose-corrected exposure at 30 min of above 40; 17were above 50, 14 were above 70; 11 were above 100; 6 were above 125;and 2 derivatives were above 150.

For comparison, compound no. 13 in Table 1 of Journal of MedicinalChemistry (2000), vol. 43, no. 9, p. 1664-669 (GLP-1(7-37) acylated atK^(26,34) with bis-C12-diacid) had a dose-corrected exposure at 30 minof below 40, and the dose-corrected exposure at 30 min for semaglutidewas in the same range of below 40.

Example 53 Effect on Blood Glucose and Body Weight

The purpose of the study is to verify the effect of the GLP-1derivatives on blood glucose (BG) and body weight (BW) in a diabeticsetting.

The GLP-1 derivatives of Examples 2, 4-5, 17, and 29 were tested in adose-response study in an obese, diabetic mouse model (db/db mice) asdescribed in the following.

Fifty db/db mice (Taconic, Denmark), fed from birth with the diet NIH31(NIH 31M Rodent Diet, commercially available from Taconic Farms, Inc.,US, see www.taconic.com), were enrolled for the study at the age of 7-9weeks The mice were given free access to standard chow (e.g. Altromin1324, Brogaarden, Gentofte, Denmark) and tap water and kept at 24° C.After 1-2 weeks of acclimatisation, the basal blood glucose was assessedtwice on two consecutive days (i.e. at 9 am). The 8 mice with the lowestblood glucose values were excluded from the experiments. Based on themean blood glucose values, the remaining 42 mice were selected forfurther experimentation and allocated to 7 groups (n=6) with matchingblood glucose levels. The mice were used in experiments with duration of5 days for up to 4 times. After the last experiment the mice wereeuthanised.

The seven groups received treatment as follows:

1: Vehicle, s.c.

2: GLP-1 derivative, 0.3 nmol/kg, s.c.

3: GLP-1 derivative, 1.0 nmol/kg, s.c.

4: GLP-1 derivative, 3.0 nmol/kg, s.c.

5: GLP-1 derivative, 10 nmol/kg, s.c.

6: GLP-1 derivative, 30 nmol/kg, s.c.

7: GLP-1 derivative, 100 nmol/kg, s.c.

Vehicle: 50 mM sodium phosphate, 145 mM sodium chloride, 0.05% Tween™80, pH 7.4.

The GLP-1 derivative was dissolved in the vehicle, to concentrations of0.05, 0.17, 0.5, 1.7, 5.0 and 17.0 nmol/ml. Animals were dosed s.c. witha dose-volume of 6 ml/kg (i.e. 300 μl per 50 g mouse).

On the day of dosing, blood glucose was assessed at time -1/2 h (8.30am), where after the mice were weighed. The GLP-1 derivative was dosedat approximately 9 am (time 0). On the day of dosing, blood glucose wasassessed at times 1, 2, 4 and 8 h (10 am, 11 am, 1 pm and 5 pm).

On the following days, the blood glucose was assessed at time 24, 48,72, and 96 h after dosing (i.e. at 9 am on day 2, 3, 4, 5). On each day,the mice were weighed following blood glucose sampling.

The mice were weighed individually on a digital weight.

Samples for the measurement of blood glucose were obtained from the tailtip capillary of conscious mice. Blood, 10 μl, was collected intoheparinised capillaries and transferred to 500 μl glucose buffer (EKFsystem solution, Eppendorf, Germany). The glucose concentration wasmeasured using the glucose oxidase method (glucose analyser Biosen 5040,EKF Diagnostic, GmbH, Barleben, Germany). The samples were kept at roomtemperature for up to 1 h until analysis. If analysis had to bepostponed, samples were kept at 4° C. for a maximum of 24 h.

ED₅₀ is the dose giving rise to half-maximal effect in nmol/kg. Thisvalue is calculated on the basis of the ability of the derivatives tolower body weight as well as the ability to lower blood glucose, asexplained below.

ED₅₀ for body weight is calculated as the dose giving rise tohalf-maximum effect on delta BW 24 hours following the subcutaneousadministration of the derivative. For example, if the maximum decreasein body weight after 24 hours is 4.0 g, then ED₅₀ bodyweight would bethat dose in nmol/kg which gives rise to a decrease in body weight after24 hours of 2.0 g. This dose (ED₅₀ body weight) may be read from thedose-response curve.

ED₅₀ for blood glucose is calcualated as the dose giving rise tohalf-maximum effect on AUC delta BG 8 hours following the subcutaneousadministration of the analogue.

The ED₅₀ value may only be calculated if a proper sigmoidaldose-response relationship exists with a clear definition of the maximumresponse. Thus, if this would not be the case the derivative in questionis re-tested in a different range of doses until the sigmoidaldose-response relationship is obtained.

The following results were obtained:

The tested derivatives had the expected effect on blood glucose as wellas on body weight (a lowering in both cases). Furthermore, a sigmoidaldose-response curve was obtained enabling the calculation of the ED₅₀values for blood glucose and body weight, respectively, as explainedabove.

Example 54 Half-Life in Minipigs

The purpose of this study is to determine the protraction in vivo of theGLP-1 derivatives after i.v. administration to minipigs, i.e. theprolongation of their time of action. This is done in a pharmacokinetic(PK) study, where the terminal half-life of the derivative in questionis determined. By terminal half-life is generally meant the period oftime it takes to halve a certain plasma concentration, measured afterthe initial distribution phase. Male Göttingen minipigs were obtainedfrom Ellegaard Göttingen Minipigs (Dalmose, Denmark) approximately 7-14months of age and weighing from approximately 16-35 kg were used in thestudies. The minipigs were housed individually and fed restrictedly onceor twice daily with SDS minipig diet (Special Diets Services, Essex,UK). After at least 2 weeks of acclimatisation two permanent centralvenous catheters were implanted in vena cava caudalis or cranialis ineach animal. The animals were allowed 1 week recovery after the surgery,and were then used for repeated pharmacokinetic studies with a suitablewash-out period between dosings.

The animals were fasted for approximately 18 h before dosing and for atleast 4 h after dosing, but had ad libitum access to water during thewhole period. The GLP-1 derivatives of Examples 2, 4-5, 16-17, 25, 29,and 39 were dissolved in 50 mM sodium phosphate, 145 mM sodium chloride,0.05% Tween™ 80, pH 7.4 to a concentration of usually from 20-60nmol/ml. Intravenous injections (the volume corresponding to usually 1-2nmol/kg, for example 0.033 ml/kg) of the compounds were given throughone catheter, and blood was sampled at predefined time points for uptill 13 days post dosing (preferably through the other catheter). Bloodsamples (for example 0.8 ml) were collected in EDTA buffer (8 mM) andthen centrifuged at 4° C. and 1942G for 10 minutes. Plasma was pippettedinto Micronic tubes on dry ice, and kept at −20° C. until analyzed forplasma concentration of the respective GLP-1 compound using ELISA or asimilar antibody based assay or LC-MS. Individual plasmaconcentration-time profiles were analyzed by a non-compartmental modelin WinNonlin v. 5.0 (Pharsight Inc., Mountain View, Calif., USA), andthe resulting terminal half-lives (harmonic mean) determined.

Results

All but one of the tested derivatives had a half-life of at least 12hours, six had a half-life of at least 24 hours, five had a half-life ofat least 36 hours, three had a half-life of at least 48 hours, and twohad a half-life of at least 60 hours.

Example 55 Effect on Glucose Mediated Insulin Secretion

The purpose of this example is to test the effect of GLP-1 derivativeson glucose mediated insulin secretion.

This is done in Göttingen minipigs using intravenous glucose tolerancetest (IVGTT).

Male Göttingen minipigs (Ellegaard Göttingen minipigs NS, Dalmose,Denmark), 7-14 months of age are used in the studies. The animals arehoused in single pens during acclimatisation and during experiments.After at least 2 weeks of acclimatisation two permanent central venouscatheters are implanted in vena cava caudalis or cranialis in eachanimal. The animals are allowed 1 week recovery after the surgery, andare then used for repeated studies with a suitable wash-out periodbetween dosings.

The pigs are fed restrictedly 1-2 times a day with SDS minipig fodder(Special Diets Services, Essex, UK) and are allowed ad libitum access towater.

The effect of the GLP-1 derivatives is tested after a single dose orafter a period with dose escalation to avoid adverse effects from acutehigh doses. The GLP-1 derivatives are given either i.v. or s.c. in thethin skin behind the ear.

For each tested GLP-1 derivative there is a vehicle treated (oruntreated) baseline group and 2-6 GLP-1 dose groups corresponding to 2-6different plasma concentration levels, which are usually from around3000-80000 pM (n=5-8).

For each GLP-1 derivative a 1 or 2 hour intravenous glucose tolerancetest is performed. The pigs are fasted for approximately 18 h before theexperiment. Patency of the central venous catheters is checked, and twobaseline blood samples are taken. After the sample at 0 minutes 0.3 g/kgglucose (Glucose 500 g/L, SAD) is given i.v. over a period of 30 secondsand the catheter is flushed with 20 ml of sterile 0.9% NaCl. Bloodsamples are usually taken at the following time points in relation tothe glucose bolus: −10, −5,0,2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70,80, 90, 100, 110, 120 minutes, and after each blood sample the catheteris flushed with 4 ml of sterile 0.9% NaCl with 10 U/ml Heparin. Bloodsamples for insulin, glucose and plasma concentrations of thederivatives are transferred to tubes coated with EDTA. The tubes arestored on wet ice until centrifugation within 1 hour (4° C., 3000 rpm,10 min), plasma is pipetted into Micronic tubes on dry ice and stored at−20° C. until analysis. Depending of the half life of the GLP-1derivative plasma concentrations are measured at t=0 min, or at t=0 minand at the end of the test (t=60 min or t=120 min). Glucose is analyzedusing the glucose oxidase method according to the manufacturer'sinstructions with 10 μL plasma in 500 μL buffer (EBIO plus autoanalyzerand solution, Eppendorf, Germany). Insulin is analyzed using a suitableimmunometric assay (such as LOCI, see e.g. Journal of BiomolecularScreening 2007, vol. 12, p. 240-247). The plasma concentration of GLP-1derivative is analyzed using ELISA or a similar antibody based assay orLC-MS.

For each study the area under the insulin curve (AUCinsulin) iscalculated and used as a measure of insulin secretion. The differentdose groups are compared to the respective vehicle/baseline group usingone-way ANOVA or other appropriate statistical analysis. An EC50 forAUCinsulin may also be calculated.

Example 56 Effect on Feed Intake

The purpose of this experiment is to investigate the effect of GLP-1derivatives on feed intake in pigs. This is done in a pharmacodynamic(PD) study as described below, in which feed intake is measured 1, 2, 3,and 4 days after administration of a single dose of the GLP-1derivative, as compared to a vehicle-treated control group.

Female Landrace Yorkshire Duroc (LYD) pigs, approximately 3 months ofage, weighing approximately 30-35 kg are used (n=3-4 per group). Theanimals are housed in a group for 1-2 weeks during acclimatisation tothe animal facilities. During the experimental period the animals areplaced in individual pens from Monday morning to Friday afternoon formeasurement of individual food intake. The animals are fed ad libitumwith pig fodder (Svinefoder, Antonio) at all times both during theacclimatisation and the experimental period. Food intake is monitored online by logging the weight of fodder every 15 minutes. The system usedis Mpigwin (Ellegaard Systems, Faaborg, Denmark).

The GLP-1 derivatives are dissolved in a phosphate buffer (50 mMphosphate, 0.05% Tween™ 80, pH 8) at concentrations of 12, 40, 120, 400or 1200 nmol/ml corresponding to doses of 0.3, 1, 3, 10 or 30 nmol/kg.The phosphate buffer served as vehicle. Animals are dosed with a singlesubcutaneous dose of the GLP-1 derivative or vehicle (dose volume 0.025ml/kg) on the morning of day 1, and feed intake is measured for 4 daysafter dosing. On the last day of each study, 4 days after dosing, ablood sample for measurement of plasma exposure of the GLP-1 derivativeis taken from the heart in anaesthetised animals. The animals arethereafter euthanised with an intra-cardial overdose of pentobarbitone.Plasma content of the GLP-1 derivatives is analysed using ELISA or asimilar antibody based assay.

Feed intake is calculated as mean±SEM 24 h food intake on the 4 days.

Statistical comparisons of the 24 hour feed intake in the vehicle vs.GLP-1 derivative group on the 4 days are done using one-way ortwo-way-ANOVA repeated measures, followed by Bonferroni post-test.

Example 57 Stability Against Degradation by Intestinal Enzymes

The purpose of this example is to test the stability against degradationby intestinal enzymes. GLP-1(7-37) was used in the assay as a kind of astandard.

All example compounds, except for the compounds of Examples 4, 6, 8,34-35, and 49, were tested.

The strongest proteolytic activities in the intestine are of pancreaticorigin and include the serine endopeptidases trypsin, chymotrypsin, andelastase as well as several types of carboxypeptidases.

An assay with small intestine extract from rats was developed and usedas described in the following.

Extracts from Rat Small Intestine

Small intestines were prepared from rats and flushed with 8 ml of 150 mMNaCl, 20 mM Hepes pH 7.4. The solutions were centrifuged for 15 min at4,600 rpm in a Heraeus Multifuge 3 S-R centrifuge with a 75006445 rotor.The supernatants were removed and filtered through a 0.22 pm MilliporeMillex GV PVDF membrane. Filtrates of several animals were pooled toaverage out individual differences.

The protein content of the obtained extracts was determined by BradfordAssay (see e.g. Analytical Biochemistry (1976), vol. 72, p. 248-254, andAnalytical Biochemistry (1996), vol. 236 p. 302-308).

Degradation Assay

2.5 nmol of the derivatives to be tested were incubated with theintestinal extract in a volume of 250 μl at 37° C. over a period of onehour. Intestinal samples were assayed in presence of 20 mM Hepes at pH7.4. The concentration of the intestinal extract was titrated in pilotexperiments so that the half-life (t½) of GLP-1(7-37) was in the rangeof 10-20 minutes. The small intestine extract was used at aconcentration of 1.4 μg/ml. All components except for the intestinalextract were mixed and pre-warmed for ten minutes at 37° C. Immediatelyafter addition of the intestinal extract a sample of 50 μl was taken andmixed with the same volume of 1% trifluoroacetic acid (TFA). Furthersamples were taken accordingly after 15, 30, and 60 minutes.

Sample Analysis

UPLC Analysis

10 μl of the samples were analysed by UPLC using a Waters Acquity systemwith a BEH C18 1.7 μm 2.1×50 mm column and a 30 to 65% gradient of 0.1%TFA and 0.07% TFA in acetonitrile over 5 minutes at a flow rate of 0.6ml/min. After baseline subtraction the peak integrals of the intactcompounds in the HPLC chromatogram recorded at a wavelength of 214 nmwere determined.

MALDI-TOF Analysis

1 μl of each sample was transferred to a Bruker/Eppendorf PAC HCCA 384MALDI target. Analysis was performed with a Bruker Autoflexmatrix-assisted laser desorption and ionisation—time of flight(MALDI-TOF) mass spectrometer using the pre-defined method “PAC_measure”with an extended detection range of 500 to 5000 Da and the pre-definedcalibration method “PAC_calibrate”.

Data Analysis

The peak integrals of the HPLC chromatograms were plotted against time.The half-life of the respective compound was calculated by fitting thedata using SigmaPlot 9.0 software and an equation for a 2-parameterexponential decay.

For each compound tested, the relative half-life (relative T_(1/2)) wascalculated as the half-life (T_(1/2)) of the compound in question,divided by the half-life (T_(1/2)) of GLP-1(7-37), determined in thesame way.

Results

The relative half-life of the known compounds liraglutide andsemaglutide was 4.8 and 1.2, respectively.

Except for one compound, all GLP-1 derivatives of the invention thatwere tested had a relative half-life of at least 1; thirty-one had arelative half-life of at least 2; and ten had a half-life of at least 5.

Example 58 Pharmacokinetics in Rat

The purpose of this Example is to investigate half-life in vivo in rat.

In vivo pharmacokinetic studies in rats were performed withten GLP-1derivatives (compounds of the present Examples 2, 4-5, 16-17, 25, 29,36, 39, and 43) of the invention, as described in the following.Semaglutide was included for comparison. Male Sprague Dawley rats ofsame age with a body weight from 400 to 600 g were obtained from Taconic(Denmark) and assigned to the treatments by simple randomisation on bodyweight, approximately 3-6 rats per group, so that all animals in eachgroup were of similar body weight.

The GLP-1 derivatives (approximately 6 nmole/ml) were dissolved in 50 mMsodium phosphate, 145 mM sodium chloride, 0.05% Tween™ 80, pH 7.4.Intravenous injections (1.0 ml/kg) of the compounds were given through acatheter implanted in the right jugular vein. Blood was sampled fromvena sublingualis for 5 days post dosing. Blood samples (200 μl) werecollected in EDTA buffer (8 mM) and then centrifuged at 4° C. and 10000Gfor 5 minutes. Plasma samples were kept at −20° C. until analyzed forplasma concentration of the respective GLP-1 compound.

The plasma concentrations of the GLP-1 compounds were determined using aLuminescence Oxygen Channeling Immunoasssay (LOCI), generally asdescribed for the determination of insulin by Poulsen and Jensen inJournal of Biomolecular Screening 2007, vol. 12, p. 240-247. The donorbeads were coated with streptavidin, while acceptor beads wereconjugated with a monoclonal antibody recognising a mid-/C-terminalepitope of the peptide. Another monoclonal antibody, specific for theN-terminus, was biotinylated. The three reactants were combined with theanalyte and formed a two-sited immuno-complex. Illumination of thecomplex released singlet oxygen atoms from the donor beads, which werechanneled into the acceptor beads and triggered chemiluminescence whichwas measured in an Envision plate reader. The amount of light wasproportional to the concentration of the compound.

Plasma concentration-time profiles were analyzed using WinNonlin (ver.5.0, Pharsight Inc., Mountain View, Calif., USA), and the half-life(T_(1/2)) calculated using individual plasma concentration-time profilesfrom each animal.

Results

The half-life of semaglutide was 4 hours.

All ten derivatives of the invention that were tested had a half-life ofat least 4 hours, all but one had a half-life of at least 8 hours, sevenhad a half-life of at least 12 hours, six had a half-life of at least 16hours, and three had a half-life of at least 24 hours.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

The invention claimed is:
 1. A derivative of a GLP-1 analogue, which analogue comprises a first K residue at a position corresponding to position 37 of GLP-1(7-37) (SEQ ID NO: 1), a second K residue at a position corresponding to position 26 of GLP-1(7-37), wherein the first K residue is designated K³⁷, and the second K residue is designated K²⁶, which analogue has a maximum of three amino acid modifications as compared to GLP-1(7-37), where two of the modifications are K³⁷ and a Q or an R at a position corresponding to position 34 of GLP-1(7-37); which derivative comprises an albumin binding moiety attached to each of K²⁶ and K³⁷ respectively, wherein the albumin binding moiety comprises a protracting moiety of formula Chem. 2a: HOOC—C₆H₄—O—(CH₂)_(y)—CO—*, in which y is an integer in the range of 7-15; or a pharmaceutically acceptable salt, amide, or ester thereof.
 2. The derivative of claim 1, wherein the analogue has two amino acid modifications as compared to GLP-1(7-37).
 3. The derivative of claim 1, wherein the analogue comprises Imp⁷ or Aib⁸.
 4. The derivative of claim 3, wherein the analogue comprises Aib⁸.
 5. The derivative of claim 4, wherein the analogue comprises the following amino acid modifications, as compared to GLP-1(7-37) (SEQ ID NO: 1): (8Aib, 34R, 37K).
 6. The derivative of claim 5, wherein the albumin binding moiety further comprises Chem. 6 and/or Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in the range of 1-5.
 7. The derivative of claim 1, wherein the albumin binding moiety further comprises Chem. 6 and/or Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in the range of 1-5.
 8. The derivative of claim 7, wherein y is
 9. 9. A compound selected from the following:

where the amino acid sequence is that of SEQ ID NO: 7,

where the amino acid sequence is that of SEQ ID NO: 7,

where the amino acid sequence is that of SEQ ID NO: 7,

where the amino acid sequence is that of SEQ ID NO: 7,

where the amino acid sequence is that of SEQ ID NO: 7,

where the amino acid sequence is that of SEQ ID NO: 12,

where the amino acid sequence is that of SEQ ID NO: 9,

where the amino acid sequence is that of SEQ ID NO: 10,

where the amino acid sequence is that of SEQ ID NO: 10,

where the amino acid sequence is that of SEQ ID NO: 8,

where the amino acid sequence is that of SEQ ID NO: 13,

where the amino acid sequence is that of SEQ ID NO: 7,

where the amino acid sequence is that of SEQ ID NO: 9,

where the amino acid sequence is that of SEQ ID NO: 9,

where the amino acid sequence is that of SEQ ID NO: 6,

where the amino acid sequence is that of SEQ ID NO: 9,

where the amino acid sequence is that of SEQ ID NO: 14,

where the amino acid sequence is that of SEQ ID NO: 7, and Chem. 68:

where the amino acid sequence is that of SEQ ID NO: 6, or a pharmaceutically acceptable salt, amide, or ester of any of these compounds.
 10. A method for treating type 2 diabetes in a subject in need of such treatment, the method comprising administering a therapeutically effective amount of a derivative according to claim 1 to a subject in need of such treatment.
 11. A method for treating type 2 diabetes in a subject in need of such treatment, the method comprising administering a therapeutically effective amount of a compound according to claim 9 to a subject in need of such treatment.
 12. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 7, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 13. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 7, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 14. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 7, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 15. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 7, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 16. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 7, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 17. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 12, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 18. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 9, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 19. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 10, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 20. A compound according to claim 1 which is

where the amino acid sequence is that of SEQ ID NO: 10, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 21. A compound according to claim 1 which is

where the amino acid sequence is that of SEQ ID NO: 8, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 22. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 13, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 23. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 7, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 24. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 9, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 25. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 9, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 26. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 6, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 27. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 9, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 28. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 14, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 29. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 7, or a pharmaceutically acceptable salt, amide, or ester of the compound.
 30. A compound according to claim 9 which is

where the amino acid sequence is that of SEQ ID NO: 6, or a pharmaceutically acceptable salt, amide, or ester of the compound. 